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Sample records for dictyostelium discoideum cells

  1. Dissection of Francisella-Host Cell Interactions in Dictyostelium discoideum.

    PubMed

    Lampe, Elisabeth O; Brenz, Yannick; Herrmann, Lydia; Repnik, Urska; Griffiths, Gareth; Zingmark, Carl; Sjöstedt, Anders; Winther-Larsen, Hanne C; Hagedorn, Monica

    2015-12-28

    Francisella bacteria cause severe disease in both vertebrates and invertebrates and include one of the most infectious human pathogens. Mammalian cell lines have mainly been used to study the mechanisms by which Francisella manipulates its host to replicate within a large variety of hosts and cell types, including macrophages. Here, we describe the establishment of a genetically and biochemically tractable infection model: the amoeba Dictyostelium discoideum combined with the fish pathogen Francisella noatunensis subsp. noatunensis. Phagocytosed F. noatunensis subsp. noatunensis interacts with the endosomal pathway and escapes further phagosomal maturation by translocating into the host cell cytosol. F. noatunensis subsp. noatunensis lacking IglC, a known virulence determinant required for Francisella intracellular replication, follows the normal phagosomal maturation and does not grow in Dictyostelium. The attenuation of the F. noatunensis subsp. noatunensis ΔiglC mutant was confirmed in a zebrafish embryo model, where growth of F. noatunensis subsp. noatunensis ΔiglC was restricted. In Dictyostelium, F. noatunensis subsp. noatunensis interacts with the autophagic machinery. The intracellular bacteria colocalize with autophagic markers, and when autophagy is impaired (Dictyostelium Δatg1), F. noatunensis subsp. noatunensis accumulates within Dictyostelium cells. Altogether, the Dictyostelium-F. noatunensis subsp. noatunensis infection model recapitulates the course of infection described in other host systems. The genetic and biochemical tractability of the system allows new approaches to elucidate the dynamic interactions between pathogenic Francisella and its host organism.

  2. Dissection of Francisella-Host Cell Interactions in Dictyostelium discoideum

    PubMed Central

    Lampe, Elisabeth O.; Brenz, Yannick; Herrmann, Lydia; Repnik, Urska; Griffiths, Gareth; Zingmark, Carl; Sjöstedt, Anders; Winther-Larsen, Hanne C.

    2015-01-01

    Francisella bacteria cause severe disease in both vertebrates and invertebrates and include one of the most infectious human pathogens. Mammalian cell lines have mainly been used to study the mechanisms by which Francisella manipulates its host to replicate within a large variety of hosts and cell types, including macrophages. Here, we describe the establishment of a genetically and biochemically tractable infection model: the amoeba Dictyostelium discoideum combined with the fish pathogen Francisella noatunensis subsp. noatunensis. Phagocytosed F. noatunensis subsp. noatunensis interacts with the endosomal pathway and escapes further phagosomal maturation by translocating into the host cell cytosol. F. noatunensis subsp. noatunensis lacking IglC, a known virulence determinant required for Francisella intracellular replication, follows the normal phagosomal maturation and does not grow in Dictyostelium. The attenuation of the F. noatunensis subsp. noatunensis ΔiglC mutant was confirmed in a zebrafish embryo model, where growth of F. noatunensis subsp. noatunensis ΔiglC was restricted. In Dictyostelium, F. noatunensis subsp. noatunensis interacts with the autophagic machinery. The intracellular bacteria colocalize with autophagic markers, and when autophagy is impaired (Dictyostelium Δatg1), F. noatunensis subsp. noatunensis accumulates within Dictyostelium cells. Altogether, the Dictyostelium-F. noatunensis subsp. noatunensis infection model recapitulates the course of infection described in other host systems. The genetic and biochemical tractability of the system allows new approaches to elucidate the dynamic interactions between pathogenic Francisella and its host organism. PMID:26712555

  3. Cell substratum adhesion during early development of Dictyostelium discoideum.

    PubMed

    Tarantola, Marco; Bae, Albert; Fuller, Danny; Bodenschatz, Eberhard; Rappel, Wouter-Jan; Loomis, William F

    2014-01-01

    Vegetative and developed amoebae of Dictyostelium discoideum gain traction and move rapidly on a wide range of substrata without forming focal adhesions. We used two independent assays to quantify cell-substrate adhesion in mutants and in wild-type cells as a function of development. Using a microfluidic device that generates a range of hydrodynamic shear stress, we found that substratum adhesion decreases at least 10 fold during the first 6 hr of development of wild type cells. This result was confirmed using a single-cell assay in which cells were attached to the cantilever of an atomic force probe and allowed to adhere to untreated glass surfaces before being retracted. Both of these assays showed that the decrease in substratum adhesion was dependent on the cAMP receptor CAR1 which triggers development. Vegetative cells missing talin as the result of a mutation in talA exhibited slightly reduced adhesive properties compared to vegetative wild-type cells. In sharp contrast to wild-type cells, however, these talA mutant cells did not show further reduction of adhesion during development such that after 5 hr of development they were significantly more adhesive than developed wild type cells. In addition, both assays showed that substrate adhesion was reduced in 0 hr cells when the actin cytoskeleton was disrupted by latrunculin. Consistent with previous observations, substrate adhesion was also reduced in 0 hr cells lacking the membrane proteins SadA or SibA as the result of mutations in sadA or sibA. However, there was no difference in the adhesion properties between wild type AX3 cells and these mutant cells after 6 hr of development, suggesting that neither SibA nor SadA play an essential role in substratum adhesion during aggregation. Our results provide a quantitative framework for further studies of cell substratum adhesion in Dictyostelium.

  4. The green tea catechin epigallocatechin gallate (EGCG) blocks cell motility, chemotaxis and development in Dictyostelium discoideum.

    PubMed

    McQuade, Kyle J; Nakajima, Akihiko; Ilacqua, April N; Shimada, Nao; Sawai, Satoshi

    2013-01-01

    Catechins, flavanols found at high levels in green tea, have received significant attention due to their potential health benefits related to cancer, autoimmunity and metabolic disease, but little is known about the mechanisms by which these compounds affect cellular behavior. Here, we assess whether the model organism Dictyostelium discoideum is a useful tool with which to characterize the effects of catechins. Epigallocatechin gallate (EGCG), the most abundant and potent catechin in green tea, has significant effects on the Dictyostelium life cycle. In the presence of EGCG aggregation is delayed, cells do not stream and development is typically stalled at the loose aggregate stage. The developmental effects very likely result from defects in motility, as EGCG reduces both random movement and chemotaxis of Dictyostelium amoebae. These results suggest that catechins and their derivatives may be useful tools with which to better understand cell motility and development in Dictyostelium and that this organism is a useful model to further characterize the activities of catechins.

  5. Detection and characterisation of NAD(P)H-diaphorase activity in Dictyostelium discoideum cells (Protozoa)

    PubMed Central

    Amaroli, A.; Chessa, M.G.

    2012-01-01

    In Dictyostelium discoideum (D. discoideum), compounds generating nitric oxide (NO) inhibit its aggregation and differentiation without altering cyclic guanosine monophosphate (cGMP) production. They do it by preventing initiation of cyclic adenosine monophosphate (cAMP) pulses. Furthermore, these compounds stimulate adenosine diphosphate (ADP)-ribosylation of a 41 kDa cytosolic protein and regulate the glyceraldehyde-3-phospate dehydrogenase activity. Yet, although D. discoideum cells produce NO at a relatively constant rate at the onset of their developmental cycle, there is still no evidence of the presence of nitric oxide synthase (NOS) enzymes. In this work, we detect the nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity in D. discoideum and we characterise it by specific inhibitors and physical-chemical conditions that allegedly distinguish between NOS-related and -unrelated NADPH-d activity. PMID:23361243

  6. Modelling cell movement, cell differentiation, cell sorting and proportion regulation in Dictyostelium discoideum aggregations.

    PubMed

    Pineda, M; Weijer, C J; Eftimie, R

    2015-04-07

    Understanding the mechanisms that control tissue morphogenesis and homeostasis is a central goal not only in developmental biology but also has great relevance for our understanding of various diseases, including cancer. A model organism that is widely used to study the control of tissue morphogenesis and proportioning is the Dictyostelium discoideum. While there are mathematical models describing the role of chemotactic cell motility in the Dictyostelium assembly and morphogenesis of multicellular tissues, as well as models addressing possible mechanisms of proportion regulation, there are no models incorporating both these key aspects of development. In this paper, we introduce a 1D hyperbolic model to investigate the role of two morphogens, DIF and cAMP, on cell movement, cell sorting, cell-type differentiation and proportioning in Dictyostelium discoideum. First, we use the non-spatial version of the model to study cell-type transdifferentiation. We perform a steady-state analysis of it and show that, depending on the shape of the differentiation rate functions, multiple steady-state solutions may occur. Then we incorporate spatial dynamics into the model, and investigate the transdifferentiation and spatial positioning of cells inside the newly formed structures, following the removal of prestalk or prespore regions of a Dictyostelium slug. We show that in isolated prespore fragments, a tipped mound-like aggregate can be formed after a transdifferentiation from prespore to prestalk cells and following the sorting of prestalk cells to the centre of the aggregate. For isolated prestalk fragments, we show the formation of a slug-like structure containing the usual anterior-posterior pattern of prestalk and prespore cells.

  7. Cellulose biogenesis in Dictyostelium discoideum

    SciTech Connect

    Blanton, R.L.

    1993-12-31

    Organisms that synthesize cellulose can be found amongst the bacteria, protistans, fungi, and animals, but it is in plants that the importance of cellulose in function (as the major structural constituent of plant cell walls) and economic use (as wood and fiber) can be best appreciated. The structure of cellulose and its biosynthesis have been the subjects of intense investigation. One of the most important insights gained from these studies is that the synthesis of cellulose by living organisms involves much more than simply the polymerization of glucose into a (1{r_arrow}4)-{beta}-linked polymer. The number of glucoses in a polymer (the degree of polymerization), the crystalline form assumed by the glucan chains when they crystallize to form a microfibril, and the dimensions and orientation of the microfibrils are all subject to cellular control. Instead of cellulose biosynthesis, a more appropriate term might be cellulose biogenesis, to emphasize the involvement of cellular structures and mechanisms in controlling polymerization and directing crystallization and deposition. Dictyostelium discoideum is uniquely suitable for the study of cellulose biogenesis because of its amenability to experimental study and manipulation and the extent of our knowledge of its basic cellular mechanisms (as will be evident from the rest of this volume). In this chapter, I will summarize what is known about cellulose biogenesis in D. discoideum, emphasizing its potential to illuminate our understanding both of D. discoideum development and plant cellulose biogenesis.

  8. Cell-cell contact mediates cAMP secretion in Dictyostelium discoideum.

    PubMed

    Fontana, D R; Price, P L; Phillips, J C

    1991-01-01

    Cyclic adenosine 3':5' monophosphate (cAMP) and cell-cell contact regulate developmental gene expression in Dictyostelium discoideum. Developing D. discoideum amoebae synthesize and secrete cAMP following the binding of cAMP to their surface cAMP receptor, a response called cAMP signaling. We have demonstrated two responses of developing D. discoideum amoebae to cell-cell contact. Cell-cell contact elicits cAMP secretion and alters the amount of cAMP secreted in a subsequent cAMP signaling response. Depending upon experimental conditions, bacterial-amoebal contact and amoebal-amoebal contact can enhance or diminish the amount of cAMP secreted during a subsequent cAMP signaling response. We have hypothesized that cell-cell contact regulates D. discoideum development by altering cellular and extracellular levels of cAMP. To begin testing this hypothesis, these responses were further characterized. The two responses to cell-cell contact are independent, i.e., they can each occur in the absence of the other. The responses to cell-cell contact also have unique temperature dependences when compared to each other, cAMP signaling, and phagocytosis. This suggests that these four responses have unique steps in their transduction mechanisms. The secretion of cAMP in response to cell-cell contact appears to be a non-specific response; contact between D. discoideum amoebae and Enterobacter aerogenes, latex beads, or other amoebae elicits cAMP secretion. Despite the apparent similarities of the effects of bacterial-amoebal and amoebal-amoebal contact on the cAMP signaling response, this contact-induced response appears to be specific. Latex beads addition does not alter the magnitude of a subsequent cAMP signaling response.(ABSTRACT TRUNCATED AT 250 WORDS)

  9. Secreted Cyclic Di-GMP Induces Stalk Cell Differentiation in the Eukaryote Dictyostelium discoideum.

    PubMed

    Chen, Zhi-hui; Schaap, Pauline

    2016-01-01

    Cyclic di-GMP (c-di-GMP) is currently recognized as the most widely used intracellular signal molecule in prokaryotes, but roles in eukaryotes were only recently discovered. In the social amoeba Dictyostelium discoideum, c-di-GMP, produced by a prokaryote-type diguanylate cyclase, induces the differentiation of stalk cells, thereby enabling the formation of spore-bearing fruiting bodies. In this review, we summarize the currently known mechanisms that control the major life cycle transitions of Dictyostelium and focus particularly on the role of c-di-GMP in stalk formation. Stalk cell differentiation has characteristics of autophagic cell death, a process that also occurs in higher eukaryotes. We discuss the respective roles of c-di-GMP and of another signal molecule, differentiation-inducing factor 1, in autophagic cell death in vitro and in stalk formation in vivo.

  10. Shear flow-induced detachment kinetics of Dictyostelium discoideum cells from solid substrate.

    PubMed Central

    Décavé, Emmanuel; Garrivier, Daniel; Bréchet, Yves; Fourcade, Bertrand; Bruckert, Franz

    2002-01-01

    Using Dictyostelium discoideum as a model organism of specific and nonspecific adhesion, we studied the kinetics of shear flow-induced cell detachment. For a given cell, detachment occurs for values of the applied hydrodynamic stress above a threshold. Cells are removed from the substrate with an apparent first-order rate constant that strongly depends on the applied stress. The threshold stress depends on cell size and physicochemical properties of the substrate, but is not affected by depolymerization of the actin and tubulin cytoskeleton. In contrast, the kinetics of cell detachment is almost independent of cell size, but is strongly affected by a modification of the substrate and the presence of an intact actin cytoskeleton. These results are interpreted in the framework of a peeling model. The threshold stress and the cell-detachment rate measure the local equilibrium energy and the dissociation rate constant of the adhesion bridges, respectively. PMID:11964228

  11. A model for individual and collective cell movement in Dictyostelium discoideum

    PubMed Central

    Palsson, Eirikur; Othmer, Hans G.

    2000-01-01

    The cellular slime mold Dictyostelium discoideum is a widely used model system for studying a variety of basic processes in development, including cell–cell signaling, signal transduction, pattern formation, cell motility, and the movement of tissue-like aggregates of cells. Many aspects of cell motion are poorly understood, including how individual cell behavior produces the collective motion of cells observed within the mound and slug. Herein, we describe a biologically realistic model for motile D. discoideum cells that can generate active forces, that interact via surface molecules, and that can detect and respond to chemotactic signals. We model the cells as deformable viscoelastic ellipsoids and incorporate signal transduction and cell–cell signaling by using a previously developed model. The shape constraint restricts the admissible deformations but makes the simulation of a large number of interacting cells feasible. Because the model is based on known processes, the parameters can be estimated or measured experimentally. We show that this model can reproduce the observations on the chemotactic behavior of single cells, streaming during aggregation, and the collective motion of an aggregate of cells driven by a small group of pacemakers. The model predicts that the motion of two-dimensional slugs [Bonner, J. T. (1998) Proc. Natl. Acad. Sci. USA 95, 9355–9359] results from the same behaviors that are exhibited by individual cells; it is not necessary to invoke different mechanisms or behaviors. Our computational experiments also suggest previously uncharacterized phenomena that may be experimentally observable. PMID:10984537

  12. Dictyostelium discoideum lipids modulate cell-cell cohesion and cyclic AMP signaling.

    PubMed Central

    Fontana, D R; Luo, C S; Phillips, J C

    1991-01-01

    During Dictyostelium discoideum development, cell-cell communication is mediated through cyclic AMP (cAMP)-induced cAMP synthesis and secretion (cAMP signaling) and cell-cell contact. Cell-cell contact elicits cAMP secretion and modulates the magnitude of a subsequent cAMP signaling response (D. R. Fontana and P. L. Price, Differentiation 41:184-192, 1989), demonstrating that cell-cell contact and cAMP signaling are not independent events. To identify components involved in the contact-mediated modulation of cAMP signaling, amoebal membranes were added to aggregation-competent amoebae in suspension. The membranes from aggregation-competent amoebae inhibited cAMP signaling at all concentrations tested, while the membranes from vegetative amoebae exhibited a concentration-dependent enhancement or inhibition of cAMP signaling. Membrane lipids inhibited cAMP signaling at all concentrations tested. The lipids abolished cAMP signaling by blocking cAMP-induced adenylyl cyclase activation. The membrane lipids also inhibited amoeba-amoeba cohesion at concentrations comparable to those which inhibited cAMP signaling. The phospholipids and neutral lipids decreased cohesion and inhibited the cAMP signaling response. The glycolipid/sulfolipid fraction enhanced cohesion and cAMP signaling. Caffeine, a known inhibitor of cAMP-induced adenylyl cyclase activation, inhibited amoeba-amoeba cohesion. These studies demonstrate that endogenous lipids are capable of modulating amoeba-amoeba cohesion and cAMP-induced activation of the adenylyl cyclase. These results suggest that cohesion may modulate cAMP-induced adenylyl cyclase activation. Because the complete elimination of cohesion is accompanied by the complete elimination of cAMP signaling, these results further suggest that cohesion may be necessary for cAMP-induced adenylyl cyclase activation in D. discoideum. PMID:1846024

  13. Aberrant spindle dynamics and cytokinesis in Dictyostelium discoideum cells that lack glycogen synthase kinase 3.

    PubMed

    Harwood, Adrian J; Forde-Thomas, Josephine E; Williams, Hazel; Samereier, Matthias; Müller-Taubenberger, Annette

    2013-01-01

    Eukaryotic cell division requires the co-ordinated assembly and disassembly of the mitotic spindle, accurate chromosome segregation and temporal control of cytokinesis to generate two daughter cells. While the absolute details of these processes differ between organisms, there are evolutionarily conserved core components common to all eukaryotic cells, whose identification will reveal the key processes that control cell division. Glycogen synthase kinase 3 (GSK-3) is a major protein kinase found throughout the eukaryotes and regulates many processes, including cell differentiation, growth, motility and apoptosis. In animals, GSK-3 associates with mitotic spindles and its inhibition causes mis-regulation of chromosome segregation. Two suppressor screens in yeast point to a more general effect of GSK-3 on cell division, however the direct role of GSK-3 in control of mitosis has not been explored outside the animal kingdom. Here we report that the Dictyostelium discoideum GSK-3 orthologue, GskA, associates with the mitotic spindle during cell division, as seen for its mammalian counterparts. Dictyostelium possesses only a single GSK-3 gene that can be deleted to eliminate all GSK-3 activity. We found that gskA-null mutants failed to elongate their mitotic spindle and were unable to divide in shaking culture, but have no chromosome segregation defect. These results suggest further conservation for the role of GSK-3 in the regulation of spindle dynamics during mitosis, but also reveal differences in the mechanisms ensuring accurate chromosome segregation.

  14. Spatiotemporal response of living cell structures in Dictyostelium discoideum with semiconductor quantum dots.

    PubMed

    Helmick, Lam; Antúnez de Mayolo, Adriana; Zhang, Ying; Cheng, Chao-Min; Watkins, Simon C; Wu, Chuanyue; LeDuc, Philip R

    2008-05-01

    The ability to monitor the spatial and temporal organization of molecules such as biopolymers within a cell is essential to enable the ability to understand the complexity and dynamics existing in biological processes. However, many limitations currently exist in specifically labeling proteins in living cells. In our study, we incorporate nanometer-sized semiconductor quantum dots (QDs) into living cells for spatiotemporal protein imaging of actin polymers in Dictyostelium discoideum without the necessity of using complicating transmembrane transport approaches. We first demonstrate cytoplasmic distribution of QDs within these living amoebae cells and then show molecular targeting through actin filament labeling. Also, we have developed a microfluidic system to control and visualize the spatiotemporal response of the cellular environment during cell motility, which allows us to demonstrate specific localization control of the QD-protein complexes in living cells. This study provides a valuable tool for the specific targeting and analysis of proteins within Dictyostelium without the encumbrance of transmembrane assisted methods, which has implication in fields including polymer physics, material science, engineering, and biology.

  15. Sentinel cells, symbiotic bacteria and toxin resistance in the social amoeba Dictyostelium discoideum.

    PubMed

    Brock, Debra A; Callison, W Éamon; Strassmann, Joan E; Queller, David C

    2016-04-27

    The social amoeba Dictyostelium discoideum is unusual among eukaryotes in having both unicellular and multicellular stages. In the multicellular stage, some cells, called sentinels, ingest toxins, waste and bacteria. The sentinel cells ultimately fall away from the back of the migrating slug, thus removing these substances from the slug. However, some D. discoideum clones (called farmers) carry commensal bacteria through the multicellular stage, while others (called non-farmers) do not. Farmers profit from their beneficial bacteria. To prevent the loss of these bacteria, we hypothesize that sentinel cell numbers may be reduced in farmers, and thus farmers may have a diminished capacity to respond to pathogenic bacteria or toxins. In support, we found that farmers have fewer sentinel cells compared with non-farmers. However, farmers produced no fewer viable spores when challenged with a toxin. These results are consistent with the beneficial bacteria Burkholderia providing protection against toxins. The farmers did not vary in spore production with and without a toxin challenge the way the non-farmers did, which suggests the costs of Burkholderia may be fixed while sentinel cells may be inducible. Therefore, the costs for non-farmers are only paid in the presence of the toxin. When the farmers were cured of their symbiotic bacteria with antibiotics, they behaved just like non-farmers in response to a toxin challenge. Thus, the advantages farmers gain from carrying bacteria include not just food and protection against competitors, but also protection against toxins.

  16. A Quorum-Sensing Factor in Vegetative Dictyostelium Discoideum Cells Revealed by Quantitative Migration Analysis

    PubMed Central

    Golé, Laurent; Rivière, Charlotte; Hayakawa, Yoshinori; Rieu, Jean-Paul

    2011-01-01

    Background Many cells communicate through the production of diffusible signaling molecules that accumulate and once a critical concentration has been reached, can activate or repress a number of target genes in a process termed quorum sensing (QS). In the social amoeba Dictyostelium discoideum, QS plays an important role during development. However little is known about its effect on cell migration especially in the growth phase. Methods and Findings To investigate the role of cell density on cell migration in the growth phase, we use multisite timelapse microscopy and automated cell tracking. This analysis reveals a high heterogeneity within a given cell population, and the necessity to use large data sets to draw reliable conclusions on cell motion. In average, motion is persistent for short periods of time (), but normal diffusive behavior is recovered over longer time periods. The persistence times are positively correlated with the migrated distances. Interestingly, the migrated distance decreases as well with cell density. The adaptation of cell migration to cell density highlights the role of a secreted quorum sensing factor (QSF) on cell migration. Using a simple model describing the balance between the rate of QSF generation and the rate of QSF dilution, we were able to gather all experimental results into a single master curve, showing a sharp cell transition between high and low motile behaviors with increasing QSF. Conclusion This study unambiguously demonstrates the central role played by QSF on amoeboid motion in the growth phase. PMID:22073217

  17. DYNAMICS OF ANTIGENIC MEMBRANE SITES RELATING TO CELL AGGREGATION IN DICTYOSTELIUM DISCOIDEUM

    PubMed Central

    Beug, H.; Katz, F. E.; Gerisch, G.

    1973-01-01

    Membrane interaction in aggregating cells of Dictyostelium discoideum can be blocked by univalent antibodies directed against specific membrane sites. Using a quantitative technique for measuring cell association, two classes of target sites for blocking antibodies were distinguished and their developmental dynamics studied. One class of these sites is specific for aggregation-competent cells, their quantity rising from virtually 0-level during growth, with a steep increase shortly before cell aggregation. The serological activity of these structures is species specific; they are not detectable in a nonaggregating mutant, but present in a revertant undergoing normal morphogenesis. Patterns of cell assembly in the presence of antibodies show that selective blockage of these membrane sites abolishes the preference for end-to-end association which is typical for aggregating cells. A second class of target sites is present in comparable quantities in particle fractions from both growth-phase and aggregation-competent cells. Blockage of these sites leads to aggregation patterns in which the side-by-side contacts of aggregating cells are abolished. The target sites of aggregation-inhibiting antibodies are suggested to be identical or associated with the molecular units of the cell membrane that mediate cell-to-cell contacts during aggregation. The results indicate that in one cell, two independent classes of contact sites can be simultaneously active. PMID:4631665

  18. Regulation of gene expression in Dictyostelium discoideum cells exposed to immobilized carbohydrates

    PubMed Central

    Bozzaro, Salvatore; Perlo, Carla; Ceccarelli, Adriano; Mangiarotti, Giorgio

    1984-01-01

    When amoebae of Dictyostelium discoideum develop on gels of polyacrylamide that are derivatized with glucosides, they become capable of aggregation at the same time as cells not exposed to glucosides. However, the aggregation centers and streams of adherent cells formed on immobilized glucosides suddenly disintegrate. The cells repeatedly re-aggregate, but never form tight aggregates as they do on other substrata. Tight aggregates formed in the absence of glucosides disperse after their transfer to glucoside gels, and the cells undergo aggregation-disaggregation cycles. The formation of tight aggregates is correlated with the expression of specific post-aggregative poly(A)+ RNAs. These RNAs are not expressed in cells developing on glucoside gels, and the dispersal of tight aggregates on such gels is accompanied by the almost complete loss of these RNAs. A developmentally regulated membrane glycoprotein called contact site A, which is a marker of aggregation-competent cells, is normally expressed on glucoside gels. Cyclic AMP is also produced, indicating that the strong increase of adenylate cyclase activity during the preaggregation phase is not affected. In conclusion, cell contact with immobilized glucosides specifically inhibits postaggregative gene expression and arrests development at the aggregation stage. ImagesFig. 1.Fig. 2.Fig. 3.Fig. 5.Fig. 7. PMID:16453493

  19. Overexpression of TOR (target of rapamycin) inhibits cell proliferation in Dictyostelium discoideum.

    PubMed

    Swer, Pynskhem Bok; Mishra, Himanshu; Lohia, Rakhee; Saran, Shweta

    2016-05-01

    TOR (target of rapamycin) protein kinase acts as a central controller of cell growth and development of an organism. Present study was undertaken to find the expression pattern and role of TOR during growth and development of Dictyostelium discoideum. Failures to generate either knockout and/or knockdown mutants indicate that interference with its levels led to cellular defects. Thus, the effects of TOR (DDB_G0281569) overexpression specifically, cells expressing Dd(Δ211-TOR)-Eyfp mutant was analyzed. Elevated expression of (Δ211-TOR)-Eyfp reduced both cell size and cell proliferation. DdTOR was found to be closer to fungus. mRNA level of TOR was found maximally in the freshly starved/aggregate cells that gradually declined. This was also strengthened by the expression patterns observed by in situ and the analysis of β-galactosidase reporter driven by the putative TOR promoter. The TOR protein was found to be highest at the aggregate stage. The fusion protein, (Δ211-TOR)-Eyfp was localized to the cell membrane, cytosol, and the nucleus. We suggest, DdTOR to be an essential protein and high TOR expression inhibits cell proliferation.

  20. Regulation of gene expression in Dictyostelium discoideum cells exposed to immobilized carbohydrates.

    PubMed

    Bozzaro, S; Perlo, C; Ceccarelli, A; Mangiarotti, G

    1984-01-01

    When amoebae of Dictyostelium discoideum develop on gels of polyacrylamide that are derivatized with glucosides, they become capable of aggregation at the same time as cells not exposed to glucosides. However, the aggregation centers and streams of adherent cells formed on immobilized glucosides suddenly disintegrate. The cells repeatedly re-aggregate, but never form tight aggregates as they do on other substrata. Tight aggregates formed in the absence of glucosides disperse after their transfer to glucoside gels, and the cells undergo aggregation-disaggregation cycles. The formation of tight aggregates is correlated with the expression of specific post-aggregative poly(A) RNAs. These RNAs are not expressed in cells developing on glucoside gels, and the dispersal of tight aggregates on such gels is accompanied by the almost complete loss of these RNAs. A developmentally regulated membrane glycoprotein called contact site A, which is a marker of aggregation-competent cells, is normally expressed on glucoside gels. Cyclic AMP is also produced, indicating that the strong increase of adenylate cyclase activity during the preaggregation phase is not affected. In conclusion, cell contact with immobilized glucosides specifically inhibits postaggregative gene expression and arrests development at the aggregation stage.

  1. Bestatin inhibits cell growth, cell division, and spore cell differentiation in Dictyostelium discoideum.

    PubMed

    Poloz, Yekaterina; Catalano, Andrew; O'Day, Danton H

    2012-04-01

    Bestatin methyl ester (BME) is an inhibitor of Zn(2+)-binding aminopeptidases that inhibits cell proliferation and induces apoptosis in normal and cancer cells. We have used Dictyostelium as a model organism to study the effects of BME. Only two Zn(2+)-binding aminopeptidases have been identified in Dictyostelium to date, puromycin-sensitive aminopeptidase A and B (PsaA and PsaB). PSA from other organisms is known to regulate cell division and differentiation. Here we show that PsaA is differentially expressed throughout growth and development of Dictyostelium, and its expression is regulated by developmental morphogens. We present evidence that BME specifically interacts with PsaA and inhibits its aminopeptidase activity. Treatment of cells with BME inhibited the rate of cell growth and the frequency of cell division in growing cells and inhibited spore cell differentiation during late development. Overexpression of PsaA-GFP (where GFP is green fluorescent protein) also inhibited spore cell differentiation but did not affect growth. Using chimeras, we have identified that nuclear versus cytoplasmic localization of PsaA affects the choice between stalk or spore cell differentiation pathway. Cells that overexpressed PsaA-GFP (primarily nuclear) differentiated into stalk cells, while cells that overexpressed PsaAΔNLS2-GFP (cytoplasmic) differentiated into spores. In conclusion, we have identified that BME inhibits cell growth, division, and differentiation in Dictyostelium likely through inhibition of PsaA.

  2. Differentiation of Dictyostelium discoideum vegetative cells into spores during Earth orbit in space.

    PubMed

    Takahashi, A; Ohnishi, K; Takahashi, S; Masukawa, M; Sekikawa, K; Amano, T; Nakano, T; Nagaoka, S; Ohnishi, T

    2001-01-01

    We reported previously that emerged amoebae of Dictyostelium (D.) discoideum grew, aggregated and differentiated to fruiting bodies with normal morphology in space. Here, we investigated the effects of space radiation and/or microgravity on the number, viability, kinetics of germination, growth rate and mutation frequency of spores formed in space in a radiation-sensitive strain, gamma s13, and the parental strain, NC4. In gamma s13, there were hardly spores in the fruiting bodies formed in space. In NC4, we found a decrease in the number of spores, a delay in germination of the spores and delayed start of cell growth of the spores formed in space when compared to the ground control. However, the mutation frequency of the NC4 spores formed in space was similar to that of the ground control. We conclude that the depression of spore formation might be induced by microgravity and/or space radiation through the depression of some stage(s) of DNA repair during cell differentiation in the slime mold.

  3. Cell behavior in Dictyostelium discoideum: preaggregation response to localized cyclic AMP pulses

    PubMed Central

    1982-01-01

    The motion of cells in the aggregation phase of Dictyostelium discoideum development is complex. To probe its mechanisms we applied precisely timed (+/- 1 s) and positioned (+/-2 micrometers) pulses of cyclic AMP to fields of cells of moderate density using a micropipette. We recorded cell behavior by time lapse microcinematography and extracted cell motion data from the film with our Galatea computer system. Analysis of these data reveals: (a) Chemotaxis lasts only about as long as the cyclic AMP signal; in particular, brief pulses (approximately 5 s) do not induce chemotaxis. (b) Chemotactic competence increases gradually from within an hour after the initiation of development (starvation) to full competence at approximately 15 h when aggregation begins under our conditions. (c) Cell motion reverses rapidly (within 20 s) when the external gradient is reversed. There is no refractory period for motion. We present a new description of the process of aggregation consistent with our result and other recent findings. (d) The behavioral response to cyclic AMP includes a phenomenon we call "cringing." In a prototypical cringe the cell speed drops within 3 s after a brief cyclic AMP stimulus, and the cell stops and rounds and then resumes motion after 25 s. (e) The development of the speed response in cringing as the cells age closely parallels the development of the cyclic AMP-induced light-scattering response of cells in suspension. (f) Cringing occurs in natural populations during weak oriented movement. The computerized analysis of cell behavior proves to be a powerful technique which can reveal significant phenomena that are not apparent to the eye even after repeated examination of the film. PMID:6282894

  4. The cytohesin paralog Sec7 of Dictyostelium discoideum is required for phagocytosis and cell motility

    PubMed Central

    2013-01-01

    Background Dictyostelium harbors several paralogous Sec7 genes that encode members of three subfamilies of the Sec7 superfamily of guanine nucleotide exchange factors. One of them is the cytohesin family represented by three members in D. discoideum, SecG, Sec7 and a further protein distinguished by several transmembrane domains. Cytohesins are characterized by a Sec7-PH tandem domain and have roles in cell adhesion and migration. Results We study here Sec7. In vitro its PH domain bound preferentially to phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2), phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). When following the distribution of GFP-Sec7 in vivo we observed the protein in the cytosol and at the plasma membrane. Strikingly, when cells formed pseudopods, macropinosomes or phagosomes, GFP-Sec7 was conspicuously absent from areas of the plasma membrane which were involved in these processes. Mutant cells lacking Sec7 exhibited an impaired phagocytosis and showed significantly reduced speed and less persistence during migration. Cellular properties associated with mammalian cytohesins like cell-cell and cell-substratum adhesion were not altered. Proteins with roles in membrane trafficking and signal transduction have been identified as putative interaction partners consistent with the data obtained from mutant analysis. Conclusions Sec7 is a cytosolic component and is associated with the plasma membrane in a pattern distinctly different from the accumulation of PI(3,4,5)P3. Mutant analysis reveals that loss of the protein affects cellular processes that involve membrane flow and the actin cytoskeleton. PMID:23915312

  5. Centromere sequence and dynamics in Dictyostelium discoideum

    PubMed Central

    Glöckner, Gernot; Heidel, Andrew J.

    2009-01-01

    Centromeres play a pivotal role in the life of a eukaryote cell, perform an essential and conserved function, but this has not led to a standard centromere structure. It remains currently unclear, how the centromeric function is achieved by widely differing structures. Since centromeres are often large and consist mainly of repetitive sequences they have only been analyzed in great detail in a handful of organisms. The genome of Dictyostelium discoideum, a valuable model organism, was described a few years ago but its centromere organization remained largely unclear. Using available sequence information we reconstructed the putative centromere organization in three of the six chromosomes of D. discoideum. They mainly consist of one type of transposons that is confined to centromeric regions. Centromeres are dynamic due to transposon integration, but an optimal centromere size seems to exist in D. discoideum. One centromere probably has expanded recently, whereas another underwent major rearrangements. In addition to insights into the centromere organization and dynamics of a protist eukaryote, this work also provides a starting point for the analysis of the evolution of centromere structures in social amoebas by comparative genomics. PMID:19179372

  6. Centromere sequence and dynamics in Dictyostelium discoideum.

    PubMed

    Glöckner, Gernot; Heidel, Andrew J

    2009-04-01

    Centromeres play a pivotal role in the life of a eukaryote cell, perform an essential and conserved function, but this has not led to a standard centromere structure. It remains currently unclear, how the centromeric function is achieved by widely differing structures. Since centromeres are often large and consist mainly of repetitive sequences they have only been analyzed in great detail in a handful of organisms. The genome of Dictyostelium discoideum, a valuable model organism, was described a few years ago but its centromere organization remained largely unclear. Using available sequence information we reconstructed the putative centromere organization in three of the six chromosomes of D. discoideum. They mainly consist of one type of transposons that is confined to centromeric regions. Centromeres are dynamic due to transposon integration, but an optimal centromere size seems to exist in D. discoideum. One centromere probably has expanded recently, whereas another underwent major rearrangements. In addition to insights into the centromere organization and dynamics of a protist eukaryote, this work also provides a starting point for the analysis of the evolution of centromere structures in social amoebas by comparative genomics.

  7. Scaling law for Dictyostelium Discoideum mounds

    NASA Astrophysics Data System (ADS)

    Voeltz, Camilla; Bodenschatz, Eberhard

    2004-03-01

    Little is known about how multicellular organisms regulate the size of their tissues during development. The eukaryote Dictyostelium Discoideum, may be studied as a model system. When starved, these amoebae aggregate and form cell mounds. These mounds develop into moving slugs and fruiting bodies consisting of a spore mass held atop a rigid stem of stalk cells. We report experiments on the development of mounds of Dicty-cells when confined to different heights. At the smallest height the amoebae are confined to a monolayer of cells in a 2d-plane. We found that the confinement inhibited the development of moving slugs and fruiting bodies. The cells aggregated and formed mounds whose size was found to be proportional to the height of the mounds. The precise mechanism is yet unknown. We will present the data and discuss possible mechanisms. This work is supported by the NSF through the Biocomplexity Program.

  8. A phototaxis signalling complex in Dictyostelium discoideum.

    PubMed

    Bandala-Sanchez, Esther; Annesley, Sarah J; Fisher, Paul R

    2006-09-01

    Phototaxis has been studied in a variety of organisms belonging to all three major taxonomic domains - the bacteria, the archaea and the eukarya. Dictyostelium discoideum is one of a small number of eukaryotic organisms which are amenable to studying the signalling pathways involved in phototaxis. In this study we provide evidence based on protein coimmunoprecipitation for a phototaxis signalling complex in Dictyostelium that includes the proteins RasD, filamin, ErkB, GRP125 and PKB.

  9. Curcumin inhibits development and cell adhesion in Dictyostelium discoideum: Implications for YakA signaling and GST enzyme function.

    PubMed

    Garige, Mamatha; Walters, Eric

    2015-11-13

    The molecular basis for nutraceutical properties of the polyphenol curcumin (Curcuma longa, Turmeric) is complex, affecting multiple factors that regulate cell signaling and homeostasis. Here, we report the effect of curcumin on cellular and developmental mechanisms in the eukaryotic model, Dictyostelium discoideum. Dictyostelium proliferation was inhibited in the presence of curcumin, which also suppressed the prestarvation marker, discoidin I, members of the yakA-mediated developmental signaling pathway, and expression of the extracellular matrix/cell adhesion proteins (DdCAD and csA). This resulted in delayed chemotaxis, adhesion, and development of the organism. In contrast to the inhibitory effects on developmental genes, curcumin induced gstA gene expression, overall GST activity, and generated production of reactive oxygen species. These studies expand our knowledge of developmental and biochemical signaling influenced by curcumin, and lends greater consideration of GST enzyme function in eukaryotic cell signaling, development, and differentiation.

  10. Chemoattractant signaling in dictyostelium discoideum.

    PubMed

    Manahan, Carol L; Iglesias, Pablo A; Long, Yu; Devreotes, Peter N

    2004-01-01

    Dictyostelium is an accessible organism for studies of signaling via chemoattractant receptors. Chemoattractant-mediated signaling events and components are reviewed and presented as a series of connected modules, including excitation, inhibition, G protein-independent responses, early gene expression, inositol lipids, PH domain-containing proteins, cyclic AMP signaling, polarization acquisition, actin polymerization, and cortical myosin. The network incorporates information from biochemical, genetic, and cell biological experiments carried out on living cells. The modules and connections represent current understanding, and future information is expected to modify and build upon this structure.

  11. Selection of chemotaxis mutants of Dictyostelium discoideum

    PubMed Central

    1987-01-01

    A method has been developed for the efficient selection of chemotaxis mutants of Dictyostelium discoideum. Mutants defective in the chemotactic response to folate could be enriched up to 30-fold in one round of selection using a chamber in which a compartment that contained the chemoattractant was separated by a sandwich of four nitrocellulose filters from a compartment that contained buffer. Mutagenized cells were placed in the center of the filter layer and exposed to the attractant gradient built up between the compartments for a period of 3-4 h. While wild-type cells moved through the filters in a wave towards the compartment that contained attractant, mutant cells remained in the filter to which they were applied. After several repetitions of the selection procedure, mutants defective in chemotaxis made up 10% of the total cell population retained in that filter. Mutants exhibiting three types of alterations were collected: motility mutants with either reduced speed of movement, or altered rates of turning; a single mutant defective in production of the attractant- degrading enzyme, folate deaminase; and mutants with normal motility but reduced chemotactic responsiveness. One mutant showed drastically reduced sensitivity in folate-induced cGMP production. Morphogenetic alterations of mutants defective in folate chemotaxis are described. PMID:3793759

  12. Developmentally regulated enzymes and cyclic AMP-binding sites in Dictyostelium discoideum cells blocked during development by alpha-chymotrypsin.

    PubMed Central

    Schmidt, J A; Stirling, J L

    1982-01-01

    When cells of the slime mould Dictyostelium discoideum are allowed to starve in the presence of alpha-chymotrypsin, they are blocked in development at the stage where tight aggregates form tips. Analysis of developmentally regulated enzymes has shown that alpha-mannosidase, beta-N-acetylglucosaminidase, threonine deaminase, tyrosine aminotransferase, beta-glucosidase and the carbohydrate-binding protein discoidin are unaffected, but enzymes that show an increase in specific activity during post-aggregative development, namely glycogen phosphorylase, UDP-glucose pyrophosphorylase, UDP-galactose 4-epimerase, UDP-galactose polysaccharide transferase and alkaline phosphatase, did not show the characteristic increase when development was blocked by alpha-chymotrypsin. Recovery of cells from the effects of alpha-chymotrypsin was accompanied by the formation of fruiting bodies and a concomitant increase in the specific activity of UDP-glucose pyrophosphorylase. Uptake or efflux of 45Ca2+ was not altered in the presence of alpha-chymotrypsin. Cells allowed to develop in alpha-chymotrypsin, or treated with the enzyme for 15 min, had a markedly reduced ability to bind cyclic AMP with low affinity; high-affinity binding was unaffected. Pronase had a similar effect on cyclic AMP binding, but trypsin, which does not alter developmental processes, has no effect on cyclic AMP binding to D. discoideum cells. PMID:7150239

  13. GPCR-controlled chemotaxis in Dictyostelium discoideum.

    PubMed

    Jin, Tian

    2011-01-01

    Dictyostelium discoideum has been chosen as the key model organism for the study of eukaryotic chemotaxis. Studies in this lower eukaryotic organism have allowed us to discover eukaryotic chemotaxis behavior and to gradually understand the mechanism of chemotaxis. Investigations in this simple organism often guide the direction of chemotaxis studies in areas such as forming concepts, discovering molecular components, revealing pathways and networks. The cooperation between experimental approaches and computational modeling has helped us to comprehend the signaling network as a system. To further reveal the relationships among the molecular mechanisms of individual signaling steps, a continuous interplay between model development and refinement and experimental testing and verification will be useful. This article focuses on a chemoattractant G-protein-coupled receptor (GPCR)/G-protein gradient sensing machinery, which is monitored by PIP(3) responses and investigated by the interplay between live cell imaging experiments and computational modeling. We believe that such an approach will lead to a much better understanding of GPCR-controlled chemotaxis of all eukaryotic cells.

  14. Effects of an extremely low-frequency electromagnetic field on stress factors: a study in Dictyostelium discoideum cells.

    PubMed

    Amaroli, Andrea; Chessa, Maria Giovanna; Bavestrello, Giorgio; Bianco, Bruno

    2013-08-01

    The development of technologies that generate environmental electromagnetic fields (EMFs) has led public opinion and the scientific community to debate upon the existence of possible effects caused by man-made EMFs on the human population and, more generally, on terrestrial ecosystems. Protozoa are known to be excellent bioassay systems in bioelectromagnetic studies because of their features that combine the reliability of in vivo results with the practicality of in vitro ones. For this reason, we examined the possible stressful effects of a 50-Hz, 300-μT extremely low-frequency electromagnetic field (ELF-EMF) on the protozoan Dictyostelium discoideum, which was used as it is included in the eight bioassay alternatives to vertebrate models for the study of human disease by the U.S. National Institutes of Health. Our results show how a 24-h exposure of D. discoideum cells to ELF-EMF can affect the net fission rate, the activity and presence of the pseudocholinesterase as well as the presence of the heat shock protein-70, while no change in the catalase and glutathione peroxidase activities was observed. However, this effect seems to be transient and all the altered parameters returned to their respective control value after a 24-h stay under dummy exposure conditions.

  15. Coupling of transcription and translation in Dictyostelium discoideum nuclei.

    PubMed

    Mangiarotti, G

    1999-03-30

    The nuclei of Dictyostelium discoideum cells have been found to contain polyribosomes active in protein synthesis. mRNA molecules enter nuclear polyribosomes while they are still being synthesized. "Non sense mediated mRNA decay" occurs in the nucleus, through the interaction of the mRNAs containing a nonsense codon with newly formed nuclear ribosomes, rather than with cytoplasmic ribosomes, as previously generally supposed.

  16. Isolation and characterization of casein kinase I from Dictyostelium discoideum.

    PubMed Central

    Moreno-Bueno, G; Calés, C; Behrens, M M; Fernández-Renart, M

    2000-01-01

    In the present study, the molecular cloning and characterization of a 49-kDa form of casein kinase (CK)I from Dictyostelium discoideum is reported. The predicted amino acid sequence shares 70% identity with the catalytic domain of the mammalian delta and epsilon isoforms, Drosophila CKIepsilon and Schizosaccharomyces pombe Hhp1, and 63% identity with Hrr25, a 57-kDa form of yeast CK involved in DNA repair. D. discoideum CKI (DdCKI) was expressed in vegetative asynchronous cells as well as in differentiated cells, as detected by Northern-blot analysis. The level of DdCKI expression did not change during the cell cycle. Antibodies raised against a truncated version of the protein recognized a 49-kDa protein from D. discoideum extracts. Protein expression paralleled the pattern found for the RNA. The expression of DdCKI in Escherichia coli resulted in an active enzyme that autophosphorylated and phosphorylated casein. Immunofluorescence assays showed that DdCKI was localized in the cytoplasm and nuclei of Dictyostelium cells. The lack of disruptants of the CKI gene suggests that this protein is essential for the vegetative growth of D. discoideum. Overexpression of DdCKI resulted in cells with increased resistance to hydroxyurea, suggesting a potential role for this kinase in DNA repair. PMID:10880352

  17. Flow-driven instabilities during pattern formation of Dictyostelium discoideum

    NASA Astrophysics Data System (ADS)

    Gholami, A.; Steinbock, O.; Zykov, V.; Bodenschatz, E.

    2015-06-01

    The slime mold Dictyostelium discoideum is a well known model system for the study of biological pattern formation. In the natural environment, aggregating populations of starving Dictyostelium discoideum cells may experience fluid flows that can profoundly change the underlying wave generation process. Here we study the effect of advection on the pattern formation in a colony of homogeneously distributed Dictyostelium discoideum cells described by the standard Martiel-Goldbeter model. The external flow advects the signaling molecule cyclic adenosine monophosphate (cAMP) downstream, while the chemotactic cells attached to the solid substrate are not transported with the flow. The evolution of small perturbations in cAMP concentrations is studied analytically in the linear regime and by corresponding numerical simulations. We show that flow can significantly influence the dynamics of the system and lead to a flow-driven instability that initiate downstream traveling cAMP waves. We also show that boundary conditions have a significant effect on the observed patterns and can lead to a new kind of instability.

  18. Mapping of a cell-binding domain in the cell adhesion molecule gp80 of Dictyostelium discoideum

    PubMed Central

    1988-01-01

    At the aggregation stage of Dictyostelium discoideum development, a cell surface glycoprotein of Mr 80,000 (gp80) has been found to mediate the EDTA-resistant type of cell-cell adhesion via homophilic interaction (Siu, C.-H., A. Cho, and A. H. C. Choi. 1987. J. Cell Biol. 105:2523-2533). To investigate the structure-function relationships of gp80, we have isolated full length cDNA clones for gp80 and determined the DNA sequence. The deduced structure of gp80 showed three major domains. An amino-terminal globular domain composed of the bulk of the protein is supported by a short stalk region, which is followed by a membrane anchor at the carboxy terminus. Structural analysis suggested that the cell-binding domain of gp80 resides within the globular domain near the amino terminus. To investigate the relationship of the cell- binding activity to this region of the polypeptide, three protein A/gp80 (PA80) gene fusions were constructed using the expression vector pRIT2T. These PA80 fusion proteins were assayed for their ability to bind to aggregation stage cells. Binding of 125I-labeled fusion proteins PA80I (containing the Val123 to Ile514 fragment of gp80) and PA80II (Val123 to Ala258) was dosage dependent and could be inhibited by precoating cells with the cell cohesion-blocking mAb 80L5C4. On the other hand, there was no appreciable binding of PA80III (Ile174 to Ile514) to cells. Reassociation of cells was significantly inhibited in the presence of PA80I or PA80II. In addition, 125I-labeled PA80II exhibited homophilic interaction with immobilized PA80I, PA80II, or gp80. The results of these studies lead to the mapping of a cell- binding domain in the region between Val123 and Leu173 of gp80 and provide direct evidence that the cell-binding activity of gp80 resides in the protein moiety. PMID:3182938

  19. Differentiation of Dictyostelium discoideum vegetative cells into spores during earth orbit in space

    NASA Astrophysics Data System (ADS)

    Takahashi, A.; Ohnishi, K.; Takahashi, S.; Masukawa, M.; Sekikawa, K.; Amano, T.; Nakano, T.; Nagaoka, S.; Ohnishi, T.

    2001-01-01

    We reported previously that emerged amoebae of Dictyosterium ( D.) discoideum grew, aggregated and differentiated to fruiting bodies with normal morphology in space. Here, we investigated the effects of space radiation and/or microgravity on the number, viability, kinetics of germination, growth rate and mutation frequency of spores formed in space in a radiation-sensitive strain, γs13, and the parental strain, NC4. In γs13, there were hardly spores in the fruiting bodies formed in space. In NC4, we found a decrease in the number of spores, a delay in germination of the spores and delayed start of cell growth of the spores formed in space when compared to the ground control. However, the mutation frequency of the NC4 spores formed in space was similar to that of the ground control. We conclude that the depression of spore formation might be induced by microgravity and/or space radiation through the depression of some stage(s) of DNA repair during cell differentiation in the slime mold.

  20. The intracellular location of lysosomal enzymes in developing Dictyostelium discoideum cells

    SciTech Connect

    Lenhard, J.M.

    1989-01-01

    The author has found that developing Dictyostelium cells contain two distinct acid hydrolase-containing organelles. Vesicles from cells at different stages of development were separated using Percoll density gradients. The lower density vesicles (LDVs or lysosomes) were present in nourished and starved cells. The higher density vesicles (HDVs) arose during starvation-induced differentiation. HDVs lacked two prestalk cell-specific lysosomal enzymes which were contained in LDVs. Prespore cell-specific spore coat proteins were detected in HDVs by ELISA. ({sup 35}S)sulfate labeling revealed that HDVs contained newly made glycoproteins as well as glycoproteins found in preexisting LDVs. Pulse-chase experiments using ({sup 35}S)methionine revealed that {alpha}-mannosidase from pre-existing LDVs an newly made {alpha}-mannosidase had entered HDVs. These data suggest that prespore LDVs mature to become HDVs. He has obtained evidence that HDVs are identical to prespore vesicles. Prespore vesicles are specialized secretory organelles which arise during prespore cell differentiation and which secrete their contents during terminal differentiation. As prespore vesicles secreted their contents, there was a co-incidental increase in extracellular acid hydrolase activity and a decrease in HDV-associated enzyme activity. Electron micrographs revealed that prespore cells contained two acid phosphatase-staining organelles, one of which appeared to be identical to lysosomes from nourished cells and a second which had features similar to prespore vesicles. Ricin-gold affinity electron microscopy was used to label the mucopolysaccharide component of prespore vesicles and the spore coat. Immunoelectron microscopy revealed co-localization of {alpha}-mannosidase with ricin-gold in prespore vesicles and the spore coat.

  1. Toxic effects of mercury on the cell nucleus of Dictyostelium discoideum.

    PubMed

    Boatti, Lara; Rapallo, Fabio; Viarengo, Aldo; Marsano, Francesco

    2017-02-01

    Governmental agencies (www.epa.gov/mercury) and the scientific community have reported on the high toxicity due to mercury. Indeed, exposure to mercury can cause severe injury to the central nervous system and kidney in humans. Beyond its recognized toxicity, little is known regarding the molecular mechanisms involved in the actions of this heavy metal. Mercury has been also observed to form insoluble fibrous protein aggregates in the cell nucleus. We used D. discoideum to evaluate micronuclei formation and, since mercury is able to induce oxidative stress that could bring to protein aggregation, we assessed nuclear protein carbonylation by Western Blot. We observed a significant increase in micronuclei formation and 14 carbonylated proteins were identified. Moreover, we used isotope-coded protein label (ICPL) and mass spectrometry analysis of proteins obtained by lysis of purified nuclei, before of tryptic digestion to quantify nuclear proteins affected by mercury. In particular, we examined the effects of mercury that associate a classical genotoxic assay to proteomic effects into the nucleus. The data present direct evidences for mercury genotoxicity, nuclear protein carbonylation, quantitative change in core histones, and the involvement of pseudouridine synthase in mercury toxicity. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 417-425, 2017.

  2. Synthesis of ribosomal proteins in developing Dictyostelium discoideum cells is controlled by the methylation of proteins S24 and S31.

    PubMed

    Mangiarotti, Giorgio

    2002-01-01

    Ribosomal protein mRNAs left over from growth are selectively excluded from polyribosomes in the first half of Dictyostelium discoideum development. This is due to the fact that they are sequestered by a class of free 40S ribosomal subunits, characterized by possessing a methylated S24 protein. At the time of formation of tight cell aggregates, the methylated S24 is substituted by an unmethylated S24, while protein S31 of the same or other 40S subunits becomes methylated. This leads to a rapid degradation of the ribosomal protein mRNAs.

  3. Analysis of Dictyostelium discoideum inositol pyrophosphate metabolism by gel electrophoresis.

    PubMed

    Pisani, Francesca; Livermore, Thomas; Rose, Giuseppina; Chubb, Jonathan Robert; Gaspari, Marco; Saiardi, Adolfo

    2014-01-01

    The social amoeba Dictyostelium discoideum was instrumental in the discovery and early characterization of inositol pyrophosphates, a class of molecules possessing highly-energetic pyrophosphate bonds. Inositol pyrophosphates regulate diverse biological processes and are attracting attention due to their ability to control energy metabolism and insulin signalling. However, inositol pyrophosphate research has been hampered by the lack of simple experimental procedures to study them. The recent development of polyacrylamide gel electrophoresis (PAGE) and simple staining to resolve and detect inositol pyrophosphate species has opened new investigative possibilities. This technology is now commonly applied to study in vitro enzymatic reactions. Here we employ PAGE technology to characterize the D. discoideum inositol pyrophosphate metabolism. Surprisingly, only three major bands are detectable after resolving acidic extract on PAGE. We have demonstrated that these three bands correspond to inositol hexakisphosphate (IP₆ or Phytic acid) and its derivative inositol pyrophosphates, IP₇ and IP₈. Biochemical analyses and genetic evidence were used to establish the genuine inositol phosphate nature of these bands. We also identified IP₉ in D. discoideum cells, a molecule so far detected only from in vitro biochemical reactions. Furthermore, we discovered that this amoeba possesses three different inositol pentakisphosphates (IP₅) isomers, which are largely metabolised to inositol pyrophosphates. Comparison of PAGE with traditional Sax-HPLC revealed an underestimation of the cellular abundance of inositol pyrophosphates by traditional methods. In fact our study revealed much higher levels of inositol pyrophosphates in D. discoideum in the vegetative state than previously detected. A three-fold increase in IP₈ was observed during development of D. discoideum a value lower that previously reported. Analysis of inositol pyrophosphate metabolism using ip6k null amoeba

  4. Chemotaxis to Excitable Waves in Dictyostelium Discoideum

    NASA Astrophysics Data System (ADS)

    Bhowmik, Arpan; Rappel, Wouter-Jan; Levine, Herbert

    In recent years, there have been significant advances in our understanding of the mechanisms underlying chemically directed motility by eukaryotic cells such as Dictyostelium. In particular, the LEGI model has proven capable of providing a framework for quantitatively explaining many experiments that present Dictyostelium cells with tailored chemical stimuli and monitor their subsequent polarization. Here, we couple the LEGI approach to an excitable medium model of the cAMP wave-field that is self-generated by the cells and investigate the extent to which this class of models enables accurate chemotaxis to the cAMP waveforms expected in vivo. Our results indicate that the ultra-sensitive version of the model does an excellent job in providing natural wave rectification, thereby providing a compelling solution to the ``back-of-the-wave paradox'' during cellular aggregation. This work was supported by National Institutes of Health Grant P01 GM078586.

  5. Comparative genomics of the social amoebae Dictyostelium discoideum and Dictyostelium purpureum

    PubMed Central

    2011-01-01

    Background The social amoebae (Dictyostelia) are a diverse group of Amoebozoa that achieve multicellularity by aggregation and undergo morphogenesis into fruiting bodies with terminally differentiated spores and stalk cells. There are four groups of dictyostelids, with the most derived being a group that contains the model species Dictyostelium discoideum. Results We have produced a draft genome sequence of another group dictyostelid, Dictyostelium purpureum, and compare it to the D. discoideum genome. The assembly (8.41 × coverage) comprises 799 scaffolds totaling 33.0 Mb, comparable to the D. discoideum genome size. Sequence comparisons suggest that these two dictyostelids shared a common ancestor approximately 400 million years ago. In spite of this divergence, most orthologs reside in small clusters of conserved synteny. Comparative analyses revealed a core set of orthologous genes that illuminate dictyostelid physiology, as well as differences in gene family content. Interesting patterns of gene conservation and divergence are also evident, suggesting function differences; some protein families, such as the histidine kinases, have undergone little functional change, whereas others, such as the polyketide synthases, have undergone extensive diversification. The abundant amino acid homopolymers encoded in both genomes are generally not found in homologous positions within proteins, so they are unlikely to derive from ancestral DNA triplet repeats. Genes involved in the social stage evolved more rapidly than others, consistent with either relaxed selection or accelerated evolution due to social conflict. Conclusions The findings from this new genome sequence and comparative analysis shed light on the biology and evolution of the Dictyostelia. PMID:21356102

  6. Sketch the migration of Dictyostelium discoideum using phase field model

    NASA Astrophysics Data System (ADS)

    Zhang, Yunsong; Camley, Brian; Rappel, Wouter-Jan; Levine, Herbert

    Cell migration plays an important role in a lot of biological processes, like chemotaxis, wound healing, and cancer metastasis. The fact it is highly integrated has brought great challenges, physical and mathematical, to the modeling efforts. Recently, a phase field model, which couples cellular reaction dynamics, intra-cellular hydrodynamics, cell-substrate adhesions and deformable cell boundaries, has successfully captured some characteristics of moving cells, including morphological change, cytosolic actin flow pattern, periodic migration and so on. Here we apply the phase field model to sketch the migration of Dictyostelium discoideum, which shows a completely different moving pattern from the cells (like fish keratocyte) in our previous attempts. And we will also compare our results with some experimental observations, not only on the cell morphology, but also on the traction force patterns on the substrate.

  7. Structural characterization of Dictyostelium discoideum prespore-specific gene D19 and of its product, cell surface glycoprotein PsA.

    PubMed Central

    Early, A E; Williams, J G; Meyer, H E; Por, S B; Smith, E; Williams, K L; Gooley, A A

    1988-01-01

    The Dictyostelium discoideum cell surface antigen PsA is a glycoprotein which first appears in the multicellular stage soon after tip formation and is selectively expressed on prespore cells. The D19 gene encodes an mRNA sequence which is highly enriched in prespore over prestalk cells in the slug stage. We have determined 81 amino acid residues of N-terminal sequence from immunoaffinity-purified PsA protein and shown this sequence to be identical to the predicted sequence of the D19 gene. There are several short repeat elements close to the C terminus, and unequal crossing-over within these is proposed to account for the size polymorphism observed in PsA protein isolated from different D. discoideum strains. The repeats are proline rich and show similarity to the C-terminal region of the D. discoideum cell adhesion molecule, contact sites A. The extreme C terminus, which is also homologous to contact sites A, is characteristic of proteins attached to the plasma membrane via a glycosyl-phosphatidylinositol link. We have marked the PsA gene by insertion of an oligonucleotide encoding an epitope of the human c-myc protein. A construct containing this gene and 990 base pairs of 5'-flanking region directed correct temporal and spatial mRNA accumulation. We found the marked PsA protein, detected with the human c-myc antibody, to be correctly localized on the surface of cells. Images PMID:2850494

  8. Excitable signal relay in Dictyostelium discoideum

    NASA Astrophysics Data System (ADS)

    Mestler, Troy; Schwab, David; Mehta, Pankaj; Gregor, Thomas

    2011-03-01

    The social amoeba D. discoideum transitions when starved from a collection of individual cells into a multicellular spore-complex. During this process, amoebae display several interesting phenomena including intercellular signaling, pattern formation, and cell differentiation. At the heart of these phenomena is the exchange of the signaling molecule cyclic-AMP, which has previously been extensively studied using a variety of indirect methods. Here we employ a sensor that uses a compound fluorescent protein whose emission spectrum changes in the presence of bound cyclic AMP to directly monitor, in real time and in vivo, intracellular cAMP concentrations. We use cells expressing this sensor in microchemostats to study intracellular cAMP concentrations at the single-cell level in response to precise, dynamically-controlled external cAMP stimulation. Specifically, we show that these cells display excitability much like that found in neurons and agree experimentally quite well with a modified FitzHugh-Nagumo dynamical systems model. This single-cell model sets groundwork for a comprehensive multicellular model that promises to explain emergent behavior in D. discoideum.

  9. Measuring cheating, fitness, and segregation in Dictyostelium discoideum.

    PubMed

    Buttery, Neil J; Smith, Jeff; Queller, David C; Strassmann, Joan E

    2013-01-01

    Dictyostelium has become a model organism for the study of social evolution because of the stage in its life cycle where thousands of independent amoebae together form a fruiting body. Some individuals die to form a stalk that holds aloft the remaining cells for dispersal to new environments as spores. Different genotypes can aggregate together, creating opportunities for exploitation by cheaters that contribute a smaller proportion of cells to the stalk. Clustering of genotypes into separate fruiting bodies reduces the opportunities for cheating. Some genotypes achieve this by segregating after aggregation. Here we describe techniques for assaying cheating and segregation in D. discoideum. We cover how to grow and maintain cells, fluorescently label genotypes, design experiments for accuracy and precision, calculate fitness and segregation, and interpret the results.

  10. Cheating by exploitation of developmental prestalk patterning in Dictyostelium discoideum.

    PubMed

    Khare, Anupama; Shaulsky, Gad

    2010-02-26

    The cooperative developmental system of the social amoeba Dictyostelium discoideum is susceptible to exploitation by cheaters-strains that make more than their fair share of spores in chimerae. Laboratory screens in Dictyostelium have shown that the genetic potential for facultative cheating is high, and field surveys have shown that cheaters are abundant in nature, but the cheating mechanisms are largely unknown. Here we describe cheater C (chtC), a strong facultative cheater mutant that cheats by affecting prestalk differentiation. The chtC gene is developmentally regulated and its mRNA becomes stalk-enriched at the end of development. chtC mutants are defective in maintaining the prestalk cell fate as some of their prestalk cells transdifferentiate into prespore cells, but that defect does not affect gross developmental morphology or sporulation efficiency. In chimerae between wild-type and chtC mutant cells, the wild-type cells preferentially give rise to prestalk cells, and the chtC mutants increase their representation in the spore mass. Mixing chtC mutants with other cell-type proportioning mutants revealed that the cheating is directly related to the prestalk-differentiation propensity of the victim. These findings illustrate that a cheater can victimize cooperative strains by exploiting an established developmental pathway.

  11. Lack of 5-methylcytosine in Dictyostelium discoideum DNA.

    PubMed Central

    Smith, S S; Ratner, D I

    1991-01-01

    We find no evidence for the presence of 5-methylcytosine in the DNA of Dictyostelium discoideum. Methylation was absent from CCGG sites in repetitive DNA and in DNA from the actin multigene family. Nor was 5-methylcytosine detected in total DNA when base composition was determined by means of h.p.l.c. Images Fig. 1. Fig. 2. PMID:1713034

  12. Secondary Ion Mass Spectrometry Imaging of Dictyostelium discoideum Aggregation Streams

    SciTech Connect

    Debord, J. Daniel; Smith, Donald F.; Anderton, Christopher R.; Heeren, Ronald M.; Pasa-Tolic, Ljiljana; Gomer, Richard H.; Fernandez-Lima, Francisco A.

    2014-06-09

    High resolution imaging mass spectrometry could become a valuable tool for cell and developmental biology, but both, high spatial and mass spectral resolution are needed to enable this. In this report, we employed Bi3 bombardment time-of-flight (Bi3 ToF-SIMS) and C60 bombardment Fourier transform ion cyclotron resonance secondary ion mass spectrometry (C60 FTICR-SIMS) to image Dictyostelium discoideum aggregation streams. Nearly 300 lipid species were identified from the aggregation streams. High resolution mass spectrometry imaging (FTICR-SIMS) enabled the generation of multiple molecular ion maps at the nominal mass level and provided good coverage for fatty acyls, prenol lipids, and sterol lipids. The comparison of Bi3 ToF-SIMS and C60 FTICR-SIMS suggested that while the first provides fast, high spatial resolution molecular ion images, the chemical complexity of biological samples warrants the use of high resolution analyzers for accurate ion identification.

  13. Crystallization of cyclase-associated protein from Dictyostelium discoideum.

    PubMed

    Hofmann, Andreas; Hess, Sonja; Noegel, Angelika A; Schleicher, Michael; Wlodawer, Alexander

    2002-10-01

    Cyclase-associated protein (CAP) is a conserved two-domain protein that helps to activate the catalytic activity of adenylyl cyclase in the cyclase-bound state through interaction with Ras, which binds to the cyclase in a different region. With its other domain, CAP can bind monomeric actin and therefore also carries a cytoskeletal function. The protein is thus involved in Ras/cAMP-dependent signal transduction and most likely serves as an adapter protein translocating the adenylyl cyclase complex to the actin cytoskeleton. Crystals belonging to the orthorhombic space group C222, with unit-cell parameters a = 71.2, b = 75.1, c = 162.9 A, have been obtained from Dictyostelium discoideum CAP carrying a C-terminal His tag. A complete native data set extending to 2.2 A resolution was collected from a single crystal using an in-house X-ray system. The asymmetric unit contains one molecule of CAP.

  14. Functional properties of five Dictyostelium discoideum P2X receptors.

    PubMed

    Baines, Abigail; Parkinson, Katie; Sim, Joan A; Bragg, Laricia; Thompson, Christopher R L; North, R Alan

    2013-07-19

    The Dictyostelium discoideum genome encodes five proteins that share weak sequence similarity with vertebrate P2X receptors. Unlike vertebrate P2X receptors, these proteins are not expressed on the surface of cells, but populate the tubules and bladders of the contractile vacuole. In this study, we expressed humanized cDNAs of P2XA, P2XB, P2XC, P2XD, and P2XE in human embryonic kidney cells and altered the ionic and proton environment in an attempt to reflect the situation in amoeba. Recording of whole-cell membrane currents showed that four receptors operated as ATP-gated channels (P2XA, P2XB, P2XD, and P2XE). At P2XA receptors, ATP was the only effective agonist of 17 structurally related putative ligands that were tested. Extracellular sodium, compared with potassium, strongly inhibited ATP responses in P2XB, P2XD, and P2XE receptors. Increasing the proton concentration (pH 6.2) accelerated desensitization at P2XA receptors and decreased currents at P2XD receptors, but increased the currents at P2XB and P2XE receptors. Dictyostelium lacking P2XA receptors showed impaired regulatory volume decrease in hypotonic solution. This phenotype was readily rescued by overexpression of P2XA and P2XD receptors, partially rescued by P2XB and P2XE receptors, and not rescued by P2XC receptors. The failure of the nonfunctional receptor P2XC to restore the regulatory volume decrease highlights the importance of ATP activation of P2X receptors for a normal response to hypo-osmotic shock, and the weak rescue by P2XB and P2XE receptors indicates that there is limited functional redundancy among Dictyostelium P2X receptors.

  15. Assessment of development and chemotaxis in Dictyostelium discoideum mutants.

    PubMed

    Artemenko, Yulia; Swaney, Kristen F; Devreotes, Peter N

    2011-01-01

    Studies using the social amoeba Dictyostelium discoideum have greatly contributed to the current understanding of the signaling network that underlies chemotaxis. Since directed migration is essential for normal D. discoideum multicellular development, mutants with chemotactic impairments are likely to have abnormal developmental morphologies. We have used multicellular development as a readout in a screen of mutants to identify new potential regulators of chemotaxis. In this chapter, we describe how mutants generated by restriction enzyme-mediated integration (REMI) are analyzed, from assessment of development to detailed characterization of 3',5'-cyclic adenosine monophosphate (cAMP)-induced responses. Two complementary approaches, plating cells either clonally on a bacterial lawn or as a population on non-nutrient agar, are used to evaluate multicellular development. Once mutants with aberrant developmental phenotypes are identified, their chemotaxis toward cAMP is assessed by both small population and micropipette assays. Furthermore, mutants are tested for defects in both general and specific signaling pathways by examining the recruitment of actin-binding LimE(Δcoil) or PIP3-binding PH domains to the plasma membrane in response to cAMP stimulation.

  16. The Long Noncoding RNA Transcriptome of Dictyostelium discoideum Development

    PubMed Central

    Rosengarten, Rafael D.; Santhanam, Balaji; Kokosar, Janez; Shaulsky, Gad

    2016-01-01

    Dictyostelium discoideum live in the soil as single cells, engulfing bacteria and growing vegetatively. Upon starvation, tens of thousands of amoebae enter a developmental program that includes aggregation, multicellular differentiation, and sporulation. Major shifts across the protein-coding transcriptome accompany these developmental changes. However, no study has presented a global survey of long noncoding RNAs (ncRNAs) in D. discoideum. To characterize the antisense and long intergenic noncoding RNA (lncRNA) transcriptome, we analyzed previously published developmental time course samples using an RNA-sequencing (RNA-seq) library preparation method that selectively depletes ribosomal RNAs (rRNAs). We detected the accumulation of transcripts for 9833 protein-coding messenger RNAs (mRNAs), 621 lncRNAs, and 162 putative antisense RNAs (asRNAs). The noncoding RNAs were interspersed throughout the genome, and were distinct in expression level, length, and nucleotide composition. The noncoding transcriptome displayed a temporal profile similar to the coding transcriptome, with stages of gradual change interspersed with larger leaps. The transcription profiles of some noncoding RNAs were strongly correlated with known differentially expressed coding RNAs, hinting at a functional role for these molecules during development. Examining the mitochondrial transcriptome, we modeled two novel antisense transcripts. We applied yet another ribosomal depletion method to a subset of the samples to better retain transfer RNA (tRNA) transcripts. We observed polymorphisms in tRNA anticodons that suggested a post-transcriptional means by which D. discoideum compensates for codons missing in the genomic complement of tRNAs. We concluded that the prevalence and characteristics of long ncRNAs indicate that these molecules are relevant to the progression of molecular and cellular phenotypes during development. PMID:27932387

  17. The Long Noncoding RNA Transcriptome of Dictyostelium discoideum Development.

    PubMed

    Rosengarten, Rafael D; Santhanam, Balaji; Kokosar, Janez; Shaulsky, Gad

    2017-02-09

    Dictyostelium discoideum live in the soil as single cells, engulfing bacteria and growing vegetatively. Upon starvation, tens of thousands of amoebae enter a developmental program that includes aggregation, multicellular differentiation, and sporulation. Major shifts across the protein-coding transcriptome accompany these developmental changes. However, no study has presented a global survey of long noncoding RNAs (ncRNAs) in D. discoideum To characterize the antisense and long intergenic noncoding RNA (lncRNA) transcriptome, we analyzed previously published developmental time course samples using an RNA-sequencing (RNA-seq) library preparation method that selectively depletes ribosomal RNAs (rRNAs). We detected the accumulation of transcripts for 9833 protein-coding messenger RNAs (mRNAs), 621 lncRNAs, and 162 putative antisense RNAs (asRNAs). The noncoding RNAs were interspersed throughout the genome, and were distinct in expression level, length, and nucleotide composition. The noncoding transcriptome displayed a temporal profile similar to the coding transcriptome, with stages of gradual change interspersed with larger leaps. The transcription profiles of some noncoding RNAs were strongly correlated with known differentially expressed coding RNAs, hinting at a functional role for these molecules during development. Examining the mitochondrial transcriptome, we modeled two novel antisense transcripts. We applied yet another ribosomal depletion method to a subset of the samples to better retain transfer RNA (tRNA) transcripts. We observed polymorphisms in tRNA anticodons that suggested a post-transcriptional means by which D. discoideum compensates for codons missing in the genomic complement of tRNAs. We concluded that the prevalence and characteristics of long ncRNAs indicate that these molecules are relevant to the progression of molecular and cellular phenotypes during development.

  18. dictyBase, the model organism database for Dictyostelium discoideum.

    PubMed

    Chisholm, Rex L; Gaudet, Pascale; Just, Eric M; Pilcher, Karen E; Fey, Petra; Merchant, Sohel N; Kibbe, Warren A

    2006-01-01

    dictyBase (http://dictybase.org) is the model organism database (MOD) for the social amoeba Dictyostelium discoideum. The unique biology and phylogenetic position of Dictyostelium offer a great opportunity to gain knowledge of processes not characterized in other organisms. The recent completion of the 34 MB genome sequence, together with the sizable scientific literature using Dictyostelium as a research organism, provided the necessary tools to create a well-annotated genome. dictyBase has leveraged software developed by the Saccharomyces Genome Database and the Generic Model Organism Database project. This has reduced the time required to develop a full-featured MOD and greatly facilitated our ability to focus on annotation and providing new functionality. We hope that manual curation of the Dictyostelium genome will facilitate the annotation of other genomes.

  19. Pattern formation in Dictyostelium discoideum aggregates in confined microenvironments

    NASA Astrophysics Data System (ADS)

    Hallou, Adrien; Hersen, Pascal; di Meglio, Jean-Marc; Kabla, Alexandre

    Dictyostelium Discoideum (Dd) is often viewed as a model system to study the complex collective cell behaviours which shape an embryo. Under starvation, Dd cells form multicellular aggregates which soon elongate, starting to display an anterior-posterior axis by differentiating into two distinct cell populations; prestalk (front) and prespore (rear) cells zones. Different models, either based on positional information or on differentiation followed up by cell sorting, have been proposed to explain the origin and the regulation of this spatial pattern.To decipher between the proposed hypotheses, we have developed am experimental platform where aggregates, made of genetically engineered Dd cells to express fluorescent reporters of cell differentiation in either prestalk or prespore cells, are allowed to develop in 20 to 400 μm wide hydrogel channels. Such a setup allows us to both mimic Dd confined natural soil environment and to follow the patterning dynamics using time-lapse microscopy. Tracking cell lineage commitments and positions in space and time, we demonstrate that Dd cells differentiate first into prestalk and prespore cells prior to sorting into an organized spatial pattern on the basis of collective motions based on differential motility and adhesion mechanisms. A. Hallou would like to thank the University of Cambridge for the Award of an ``Oliver Gatty Studentship in Biophysical and Colloid Science''.

  20. Quantitative analysis of cell motility and chemotaxis in Dictyostelium discoideum by using an image processing system and a novel chemotaxis chamber providing stationary chemical gradients

    PubMed Central

    1989-01-01

    An image processing system was programmed to automatically track and digitize the movement of amebae under phase-contrast microscopy. The amebae moved in a novel chemotaxis chamber designed to provide stable linear attractant gradients in a thin agarose gel. The gradients were established by pumping attractant and buffer solutions through semipermeable hollow fibers embedded in the agarose gel. Gradients were established within 30 min and shown to be stable for at least a further 90 min. By using this system it is possible to collect detailed data on the movement of large numbers of individual amebae in defined attractant gradients. We used the system to study motility and chemotaxis by a score of Dictyostelium discoideum wild-type and mutant strains, including "streamer" mutants which are generally regarded as being altered in chemotaxis. None of the mutants were altered in chemotaxis in the optimal cAMP gradient of 25 nM/mm, with a midpoint of 25 nM. The dependence of chemotaxis on cAMP concentration, gradient steepness, and temporal changes in the gradient were investigated. We also analyzed the relationship between turning behavior and the direction of travel during chemotaxis in stable gradients. The results suggest that during chemotaxis D. discoideum amebae spatially integrate information about local increases in cAMP concentration at various points on the cell surface. PMID:2537839

  1. Dictyostelium cell death

    PubMed Central

    Levraud, Jean-Pierre; Adam, Myriam; Luciani, Marie-Françoise; de Chastellier, Chantal; Blanton, Richard L.; Golstein, Pierre

    2003-01-01

    Cell death in the stalk of Dictyostelium discoideum, a prototypic vacuolar cell death, can be studied in vitro using cells differentiating as a monolayer. To identify early events, we examined potentially dying cells at a time when the classical signs of Dictyostelium cell death, such as heavy vacuolization and membrane lesions, were not yet apparent. We observed that most cells proceeded through a stereotyped series of differentiation stages, including the emergence of “paddle” cells showing high motility and strikingly marked subcellular compartmentalization with actin segregation. Paddle cell emergence and subsequent demise with paddle-to-round cell transition may be critical to the cell death process, as they were contemporary with irreversibility assessed through time-lapse videos and clonogenicity tests. Paddle cell demise was not related to formation of the cellulose shell because cells where the cellulose-synthase gene had been inactivated underwent death indistinguishable from that of parental cells. A major subcellular alteration at the paddle-to-round cell transition was the disappearance of F-actin. The Dictyostelium vacuolar cell death pathway thus does not require cellulose synthesis and includes early actin rearrangements (F-actin segregation, then depolymerization), contemporary with irreversibility, corresponding to the emergence and demise of highly polarized paddle cells. PMID:12654899

  2. Identification and characterization of a Dictyostelium discoideum ribosomal protein gene.

    PubMed Central

    Szymkowski, D E; Deering, R A

    1990-01-01

    We have identified a developmentally repressed large-subunit ribosomal protein gene of Dictyostelium discoideum based on sequence similarity to other ribosomal proteins. Protein rpl7 is homologous to large subunit ribosomal proteins from the rat and possibly to Mycoplasma capricolum and Escherichia coli, but is not similar to three sequenced ribosomal proteins in Dictyostelium. The rpl7 gene is present at one copy per genome, as are six other cloned Dictyostelium ribosomal proteins. Restriction fragment length polymorphisms exist for ribosomal protein genes rpl7, rp1024, and rp110 in strain HU182; most Dictyostelium ribosomal protein genes examined are linked no closer than 30-100 kb to each other in the genome. Dictyostelium ribosomal proteins are known to be developmentally regulated, and levels of rpl7 transcript gradually decrease during the 24-hour development cycle. This drop correlates with that of rp1024, indicating these and other ribosomal protein genes may be coordinately regulated. To determine the cellular location of the protein, we raised antibodies to an rpl7-derived branched synthetic peptide. These antibodies cross-reacted with one protein of the expected size in a ribosomal protein fraction of Dictyostelium, indicating that the product of gene rpl7 is localized in the ribosome. Images PMID:1975664

  3. AmpA protein functions by different mechanisms to influence early cell type specification and to modulate cell adhesion and actin polymerization in Dictyostelium discoideum.

    PubMed

    Cost, Hoa N; Noratel, Elizabeth F; Blumberg, Daphne D

    2013-01-01

    The Dictyostelium discoideum ampA gene encodes a multifunctional regulator protein that modulates cell-cell and cell-substrate adhesions and actin polymerization during growth and is necessary for correct cell type specification and patterning during development. Insertional inactivation of the ampA gene results in defects that define two distinct roles for the ampA gene during development. AmpA is necessary in a non-cell autonomous manner to prevent premature expression of a prespore gene marker. It is also necessary in a cell autonomous manner for the anterior like cells, which express the ampA gene, to migrate to the upper cup during culmination. It is also necessary to prevent excessive cell-cell agglutination when cells are developed in a submerged suspension culture. Here, we demonstrate that a supernatant source of AmpA protein, added extracellularly, can prevent the premature mis-expression of the prespore marker. Synthetic oligopeptides are used to identify the domain of the AmpA protein that is important for preventing cells from mis-expressing the prespore gene. We further demonstrate that a factor capable of inducing additional cells to express the prespore gene marker accumulates extracellularly in the absence of AmpA protein. While the secreted AmpA acts extracellularly to suppress prespore gene expression, the effects of AmpA on cell agglutination and on actin polymerization in growing cells are not due to an extracellular role of secreted AmpA protein. Rather, these effects appear to reflect a distinct cell autonomous role of the ampA gene. Finally, we show that secretion of AmpA protein is brought about by elevating the levels of expression of ampA so that the protein accumulates to an excessive level.

  4. Dictyostelium discoideum: a new host model system for intracellular pathogens of the genus Legionella.

    PubMed

    Hägele, S; Köhler, R; Merkert, H; Schleicher, M; Hacker, J; Steinert, M

    2000-04-01

    The soil amoeba Dictyostelium discoideum is a haploid eukaryote that, upon starvation, aggregates and enters a developmental cycle to produce fruiting bodies. In this study, we infected single-cell stages of D. discoideum with different Legionella species. Intracellular growth of Legionella in this new host system was compared with their growth in the natural host Acanthamoeba castellanii. Transmission electron microscopy of infected D. discoideum cells revealed that legionellae reside within the phagosome. Using confocal microscopy, it was observed that replicating, intracellular, green fluorescent protein (GFP)-tagged legionellae rarely co-localized with fluorescent antibodies directed against the lysosomal protein DdLIMP of D. discoideum. This indicates that the bacteria inhibit the fusion of phagosomes and lysosomes in this particular host system. In addition, Legionella infection of D. discoideum inhibited the differentiation of the host into the multicellular fruiting stage. Co-culture studies with profilin-minus D. discoideum mutants and Legionella resulted in higher rates of infection when compared with infections of wild-type amoebae. Because the amoebae are amenable to genetic manipulation as a result of their haploid genome and because a number of cellular markers are available, we show for the first time that D. discoideum is a valuable model system for studying intracellular pathogenesis of microbial pathogens.

  5. Scanning X-Ray Nanodiffraction on Dictyostelium discoideum

    PubMed Central

    Priebe, Marius; Bernhardt, Marten; Blum, Christoph; Tarantola, Marco; Bodenschatz, Eberhard; Salditt, Tim

    2014-01-01

    We have performed scanning x-ray nanobeam diffraction experiments on single cells of the amoeba Dictyostelium discoideum. Cells have been investigated in 1), freeze-dried, 2), frozen-hydrated (vitrified), and 3), initially alive states. The spatially resolved small-angle x-ray scattering signal shows characteristic streaklike patterns in reciprocal space, which we attribute to fiber bundles of the actomyosin network. From the intensity distributions, an anisotropy parameter can be derived that indicates pronounced local variations within the cell. In addition to nanobeam small-angle x-ray scattering, we have evaluated the x-ray differential phase contrast in view of the projected electron density. Different experimental aspects of the x-ray experiment, sample preparation, and data analysis are discussed. Finally, the x-ray results are correlated with optical microscopy (differential phase contrast and confocal microscopy of mutant strains with fluorescently labeled actin and myosin II), which have been carried out in live and fixed states, including optical microscopy under cryogenic conditions. PMID:25468345

  6. Dictyostelium discoideum as a Novel Host System to Study the Interaction between Phagocytes and Yeasts

    PubMed Central

    Koller, Barbara; Schramm, Christin; Siebert, Susann; Triebel, János; Deland, Eric; Pfefferkorn, Anna M.; Rickerts, Volker; Thewes, Sascha

    2016-01-01

    The social amoeba Dictyostelium discoideum is a well-established model organism to study the interaction between bacteria and phagocytes. In contrast, research using D. discoideum as a host model for fungi is rare. We describe a comprehensive study, which uses D. discoideum as a host model system to investigate the interaction with apathogenic (Saccharomyces cerevisiae) and pathogenic (Candida sp.) yeast. We show that Dictyostelium can be co-cultivated with yeasts on solid media, offering a convenient test to study the interaction between fungi and phagocytes. We demonstrate that a number of D. discoideum mutants increase (atg1−, kil1−, kil2−) or decrease (atg6−) the ability of the amoebae to predate yeast cells. On the yeast side, growth characteristics, reduced phagocytosis rate, as well as known virulence factors of C. albicans (EFG1, CPH1, HGC1, ICL1) contribute to the resistance of yeast cells against predation by the amoebae. Investigating haploid C. albicans strains, we suggest using the amoebae plate test for screening purposes after random mutagenesis. Finally, we discuss the potential of our adapted amoebae plate test to use D. discoideum for risk assessment of yeast strains. PMID:27818653

  7. Evidence for the presence of an NF-kappaB signal transduction system in Dictyostelium discoideum.

    PubMed

    Traincard, F; Ponte, E; Pun, J; Coukell, B; Veron, M

    1999-10-01

    The Rel/NF-kappaB family of transcription factors and regulators has so far only been described in vertebrates and arthropods, where they mediate responses to many extracellular signals. No counterparts of genes coding for such proteins have been identified in the Caenorhabditis elegans genome and no NF-kappaB activity was found in Saccharomyces cerevisiae. We describe here the presence of an NF-kappaB transduction pathway in the lower eukaryote Dictyostelium discoideum. Using antibodies raised against components of the mammalian NF-kappaB pathway, we demonstrate in Dictyostelium cells extracts the presence of proteins homologous to Rel/NF-kappaB, IkappaB and IKK components. Using gel-shift experiments in nuclear extracts of developing Dictyostelium cells, we demonstrate the presence of proteins binding to kappaB consensus oligonucleotides and to a GC-rich kappaB-like sequence, lying in the promoter of cbpA, a developmentally regulated Dictyostelium gene encoding the Ca(2+)-binding protein CBP1. Using immunofluorescence, we show specific nuclear translocation of the p65 and p50 homologues of the NF-kappaB transcription factors as vegetatively growing cells develop to the slug stage. Taken together, our results strongly indicate the presence of a complete NF-kappaB signal transduction system in Dictyostelium discoideum that could be involved in the developmental process.

  8. The use of streptavidin conjugates as immunoblot loading controls and mitochondrial markers for use with Dictyostelium discoideum.

    PubMed

    Davidson, Andrew J; King, Jason S; Insall, Robert H

    2013-07-01

    The loading controls used for quantitative immunoblotting of mammalian proteins are not appropriate for use with Dictyostelium discoideum. Actin levels, for example, change greatly during Dictyostelium development. In addition, Dictyostelium-specific antibodies for other potential control proteins are not commercially available. Here we demonstrate the use of labeled streptavidin to detect biotinylated mitochondrial 3-methylcrotonyl-CoA carboxylase α (MCCC1), providing a robust and convenient tool for quantitative normalization of Dictyostelium Western blots, as well as fluorescently labeling mitochondria for microscopy of fixed cells.

  9. TgrC1 mediates cell-cell adhesion by interacting with TgrB1 via mutual IPT/TIG domains during development of Dictyostelium discoideum.

    PubMed

    Chen, Gong; Wang, Jun; Xu, Xiaoqun; Wu, Xiangfu; Piao, Ruihan; Siu, Chi-Hung

    2013-06-01

    Cell-cell adhesion plays crucial roles in cell differentiation and morphogenesis during development of Dictyostelium discoideum. The heterophilic adhesion protein TgrC1 (Tgr is transmembrane, IPT, IG, E-set, repeat protein) is expressed during cell aggregation, and disruption of the tgrC1 gene results in the arrest of development at the loose aggregate stage. We have used far-Western blotting coupled with MS to identify TgrB1 as the heterophilic binding partner of TgrC1. Co-immunoprecipitation and pull-down studies showed that TgrB1 and TgrC1 are capable of binding with each other in solution. TgrB1 and TgrC1 are encoded by a pair of adjacent genes which share a common promoter. Both TgrB1 and TgrC1 are type I transmembrane proteins, which contain three extracellular IPT/TIG (immunoglobulin, plexin, transcription factor-like/transcription factor immunoglobulin) domains. Antibodies raised against TgrB1 inhibit cell reassociation at the post-aggregation stage of development and block fruiting body formation. Ectopic expression of TgrB1 and TgrC1 driven by the actin15 promoter leads to heterotypic cell aggregation of vegetative cells. Using recombinant proteins that cover different portions of TgrB1 and TgrC1 in binding assays, we have mapped the cell-binding regions in these two proteins to Lys(537)-Ala(783) in TgrB1 and Ile(336)-Val(360) in TgrC1, corresponding to their respective TIG3 and TIG2 domain.

  10. Lipopolysaccharide induction of autophagy is associated with enhanced bactericidal activity in Dictyostelium discoideum

    PubMed Central

    Pflaum, Katherine; Gerdes, Kimberly; Yovo, Kossi; Callahan, Jennifer; Snyder, Michelle L.D.

    2012-01-01

    Innate immune cells respond to microbial invaders using pattern recognition receptors that detect conserved microbial patterns. Among the cellular processes stimulated downstream of pattern recognition machinery is the initiation of autophagy, which plays protective roles against intracellular microbes. We have shown recently that Dictyostelium discoideum, which takes up bacteria for nutritive purposes, may employ pattern recognition machinery to respond to bacterial prey, as D. discoideum cells upregulate bactericidal activity upon stimulation by lipopolysaccharide (LPS). Here we extend these findings, showing that LPS treatment leads to induction of autophagosomal maturation in cells responding to the bacteria Staphylococcus aureus. Cells treated with the autophagy-inducing drug rapamycin clear internalized bacteria at an accelerated rate, while LPS-enhanced clearance of bacteria is reduced in cells deficient for the autophagy-related genes atg1 and atg9. These findings link microbial pattern recognition with autophagy in the social amoeba D. discoideum. PMID:22575510

  11. Cell type specificity and mechanism of control of a gene may be reverted in different strains of Dictyostelium discoideum.

    PubMed

    Mangiarotti, G; Giorda, R

    2000-06-21

    Twelve genes which are expressed exclusively in pre-spore cells of Dictyostelium strain AX3 are expressed exclusively in pre-stalk cells of strain AX2. One gene has the opposite behavior: it is expressed in pre-stalk cells in AX3 and in pre-spore cells in AX2. The change in cell type specificity involves a change in the mechanism of control of gene expression. When they are expressed in pre-stalk cells, genes are controlled at the level of transcription, whilst in pre-spore cells, they are controlled at the level of mRNA stability. Genes expressed in pre-stalk cells in strain AX2, fused with an AX2 pre-spore specific promoter, become regulated at the level of mRNA stability. These findings indicate that at least a group of pre-stalk mRNAs possess the cis-destabilizing element typical of pre-spore mRNAs, though they are not destabilized in disaggregated cells. This is due to the fact that ribosomal protein S6, phosphorylation of which is responsible for controlling the stability of pre-spore mRNAs, is not dephosphorylated in disaggregated pre-stalk cells. These cells lack an S6 phosphatase activity which has been purified from disaggregated pre-spore cells.

  12. The cell adhesion molecule DdCAD-1 regulates morphogenesis through differential spatiotemporal expression in Dictyostelium discoideum.

    PubMed

    Sriskanthadevan, Shrivani; Zhu, Yingyue; Manoharan, Kumararaaj; Yang, Chunxia; Siu, Chi-Hung

    2011-06-01

    During development of Dictyostelium, multiple cell types are formed and undergo a coordinated series of morphogenetic movements guided by their adhesive properties and other cellular factors. DdCAD-1 is a unique homophilic cell adhesion molecule encoded by the cadA gene. It is synthesized in the cytoplasm and transported to the plasma membrane by contractile vacuoles. In chimeras developed on soil plates, DdCAD-1-expressing cells showed greater propensity to develop into spores than did cadA-null cells. When development was performed on non-nutrient agar, wild-type cells sorted from the cadA-null cells and moved to the anterior zone. They differentiated mostly into stalk cells and eventually died, whereas the cadA-null cells survived as spores. To assess the role of DdCAD-1 in this novel behavior of wild-type and mutant cells, cadA-null cells were rescued by the ectopic expression of DdCAD-1-GFP. Morphological studies have revealed major spatiotemporal changes in the subcellular distribution of DdCAD-1 during development. Whereas DdCAD-1 became internalized in most cells in the post-aggregation stages, it was prominent in the contact regions of anterior cells. Cell sorting was also restored in cadA(-) slugs by exogenous recombinant DdCAD-1. Remarkably, DdCAD-1 remained on the surface of anterior cells, whereas it was internalized in the posterior cells. Additionally, DdCAD-1-expressing cells migrated slower than cadA(-) cells and sorted to the anterior region of chimeric slugs. These results show that DdCAD-1 influences the sorting behavior of cells in slugs by its differential distribution on the prestalk and prespore cells.

  13. Mitochondrial large-conductance potassium channel from Dictyostelium discoideum.

    PubMed

    Laskowski, Michal; Kicinska, Anna; Szewczyk, Adam; Jarmuszkiewicz, Wieslawa

    2015-03-01

    In the present study, we describe the existence of a large-conductance calcium-activated potassium (BKCa) channel in the mitochondria of Dictyostelium discoideum. A single-channel current was recorded in a reconstituted system, using planar lipid bilayers. The large-conductance potassium channel activity of 258±12 pS was recorded in a 50/150 mM KCl gradient solution. The probability of channel opening (the channel activity) was increased by calcium ions and NS1619 (potassium channel opener) and reduced by iberiotoxin (BKCa channel inhibitor). The substances known to modulate BKCa channel activity influenced the bioenergetics of D. discoideum mitochondria. In isolated mitochondria, NS1619 and NS11021 stimulated non-phosphorylating respiration and depolarized membrane potential, indicating the channel activation. These effects were blocked by iberiotoxin and paxilline. Moreover, the activation of the channel resulted in attenuation of superoxide formation, but its inhibition had the opposite effect. Immunological analysis with antibodies raised against mammalian BKCa channel subunits detected a pore-forming α subunit and auxiliary β subunits of the channel in D. discoideum mitochondria. In conclusion, we show for the first time that mitochondria of D. discoideum, a unicellular ameboid protozoon that facultatively forms multicellular structures, contain a large-conductance calcium-activated potassium channel with electrophysiological, biochemical and molecular properties similar to those of the channels previously described in mammalian and plant mitochondria.

  14. Spore germination promoter of Dictyostelium discoideum excreted by Aerobacter aerogenes.

    PubMed

    Hashimoto, Y; Tanaka, Y; Yamada, T

    1976-07-01

    The nutrient medium in which Aerobacter aerogenes was grown, contains a spore germination promoter (SGP) for the cellular slime mould Dictyostelium discoideum. SGP can cuase synchronous spore germination in a short time, and triggers the germination process in just a few minutes. Germination-promoting capacity of SGP decreases as it comes in contact with increasing number of spores. When spores activated by SGP are stored at 4 degrees C, they gradually return to the dormant state. SGP is comparatively heat-stable, but is unstable at pH above 10 or under 3.

  15. Ground Testing of the EMCS Seed Cassette for Biocompatibility with the Cellular Slime Mold, Dictyostelium Discoideum

    NASA Technical Reports Server (NTRS)

    Hanely, Julia C.; Reinsch, Sigrid; Myers, Zachary A.; Freeman, John; Steele, Marianne K.; Sun, Gwo-Shing; Heathcote, David G.

    2014-01-01

    The European Modular Cultivation System, EMCS, was developed by ESA for plant experiments. To expand the use of flight verified hardware for various model organisms, we performed ground experiments to determine whether ARC EMCS Seed Cassettes could be adapted for use with cellular slime mold for future space flight experiments. Dictyostelium is a cellular slime mold that can exist both as a single-celled independent organism and as a part of a multicellular colony which functions as a unit (pseudoplasmodium). Under certain stress conditions, individual amoebae will aggregate to form multicellular structures. Developmental pathways are very similar to those found in Eukaryotic organisms, making this a uniquely interesting organism for use in genetic studies. Dictyostelium has been used as a genetic model organism for prior space flight experiments. Due to the formation of spores that are resistant to unfavorable conditions such as desiccation, Dictyostelium is also a good candidate for use in the EMCS Seed Cassettes. The growth substratum in the cassettes is a gridded polyether sulfone (PES) membrane. A blotter beneath the PES membranes contains dried growth medium. The goals of this study were to (1) verify that Dictyostelium are capable of normal growth and development on PES membranes, (2) develop a method for dehydration of Dictyostelium spores with successful recovery and development after rehydration, and (3) successful mock rehydration experiments in cassettes. Our results show normal developmental progression in two strains of Dictyostelium discoideum on PES membranes with a bacterial food source. We have successfully performed a mock rehydration of spores with developmental progression from aggregation to slug formation, and production of morphologically normal spores within 9 days of rehydration. Our results indicate that experiments on the ISS using the slime mold, Dictyostelium discoideum could potentially be performed in the flight verified hardware of

  16. The cyclin-dependent kinase family in the social amoebozoan Dictyostelium discoideum.

    PubMed

    Huber, Robert J

    2014-02-01

    Cyclin-dependent kinases (Cdk) are a family of serine/threonine protein kinases that regulate eukaryotic cell cycle progression. Their ability to modulate the cell cycle has made them an attractive target for anti-cancer therapies. Cdk protein function has been studied in a variety of Eukaryotes ranging from yeast to humans. In the social amoebozoan Dictyostelium discoideum, several homologues of mammalian Cdks have been identified and characterized. The life cycle of this model organism is comprised of a feeding stage where single cells grow and divide mitotically as they feed on their bacterial food source and a multicellular developmental stage that is induced by starvation. Thus it is a valuable system for studying a variety of cellular and developmental processes. In this review I summarize the current knowledge of the Cdk protein family in Dictyostelium by highlighting the research efforts focused on the characterization of Cdk1, Cdk5, and Cdk8 in this model Eukaryote. Accumulated evidence indicates that each protein performs distinct functions during the Dictyostelium life cycle with Cdk1 being required for growth and Cdk5 and Cdk8 being required for processes that occur during development. Recent studies have shown that Dictyostelium Cdk5 shares attributes with mammalian Cdk5 and that the mammalian Cdk inhibitor roscovitine can be used to inhibit Cdk5 activity in Dictyostelium. Together, these results show that Dictyostelium can be used as a model system for studying Cdk protein function.

  17. Synthesis and SAR of 4-methyl-5-pentylbenzene-1,3-diol (MPBD), produced by Dictyostelium discoideum.

    PubMed

    Murata, Chihiro; Ogura, Tetsuhiro; Narita, Shuhei; Kondo, Anna P; Iwasaki, Natsumi; Saito, Tamao; Usuki, Toyonobu

    2016-03-01

    4-Methyl-5-pentylbenzene-1,3-diol (MPBD) is a secondary metabolite of SteelyA polyketide synthase, which controls cell aggregation and spore maturation of Dictyostelium discoideum. In this study, chemical synthesis of MPBD and its derivatives was achieved. Structure-activity relationship (SAR) studies for antimicrobial activities against Escherichia coli and Bacillus subtilis were also conducted.

  18. Migration in the social stage of Dictyostelium discoideum amoebae impacts competition

    PubMed Central

    Buttery, Neil; Adu-Oppong, Boahemaa; Powers, Michael; Thompson, Christopher R.L.; Queller, David C.; Strassmann, Joan E.

    2015-01-01

    Interaction conditions can change the balance of cooperation and conflict in multicellular groups. After aggregating together, cells of the social amoeba Dictyostelium discoideum may migrate as a group (known as a slug) to a new location. We consider this migration stage as an arena for social competition and conflict because the cells in the slug may not be from a genetically homogeneous population. In this study, we examined the interplay of two seemingly diametric actions, the solitary action of kin recognition and the collective action of slug migration in D. discoideum, to more fully understand the effects of social competition on fitness over the entire lifecycle. We compare slugs composed of either genetically homogenous or heterogeneous cells that have migrated or remained stationary in the social stage of the social amoeba Dictyostelium discoideum. After migration of chimeric slugs, we found that facultative cheating is reduced, where facultative cheating is defined as greater contribution to spore relative to stalk than found for that clone in the clonal state. In addition our results support previous findings that competitive interactions in chimeras diminish slug migration distance. Furthermore, fruiting bodies have shorter stalks after migration, even accounting for cell numbers at that time. Taken together, these results show that migration can alleviate the conflict of interests in heterogeneous slugs. It aligns their interest in finding a more advantageous place for dispersal, where shorter stalks suffice, which leads to a decrease in cheating behavior. PMID:26528414

  19. Studies on the transcription, translation, and structure of alpha-actinin in Dictyostelium discoideum.

    PubMed

    Witke, W; Schleicher, M; Lottspeich, F; Noegel, A

    1986-09-01

    A clone coding for the F-actin cross-linking protein alpha-actinin was obtained by screening a genomic library of Dictyostelium discoideum DNA in lambda gt11 with monoclonal antibodies specific for Dictyostelium alpha-actinin. The 1.2-kilobase (kb) genomic clone was confirmed as containing part of the alpha-actinin gene by comparing its nucleotide sequence with the amino acid sequence of tryptic peptides from purified alpha-actinin. The clone recognized a 3.0-kb message in a Northern blot. Hybridization to RNA isolated from different developmental stages of several D. discoideum strains indicated that the mRNA content increased during early development. A similar result was obtained when the alpha-actinin content of the cells was followed by Western blot analysis. Hybridization of the clone to DNA from different wild-type strains of D. discoideum indicated a polymorphism on the DNA level that coincided with a polymorphism on the protein level. The data suggest continuous transcription of the alpha-actinin gene throughout the development of D. discoideum, up- and down-regulation of the levels of alpha-actinin mRNA and protein with maximum levels at the onset of aggregation, and a high diversity of alpha-actinin at the DNA and protein level among different D. discoideum strains. The structural data make it conceivable that the highly conserved nature of alpha-actinin resides only at the functional sites, whereas the helical portions of the alpha-actinin molecule allow a higher level of diversity throughout evolution.

  20. A neutral ceramidase homologue from Dictyostelium discoideum exhibits an acidic pH optimum.

    PubMed Central

    Monjusho, Hatsumi; Okino, Nozomu; Tani, Motohiro; Maeda, Mineko; Yoshida, Motonobu; Ito, Makoto

    2003-01-01

    The nucleotide sequence reported for the Dictyostelium discoideum ceramidase is available on the DNA Data Bank of Japan (DDBJ). Ceramidases (CDases) are currently classified into three categories (acid, neutral and alkaline) based on their optimal pHs and primary structures. Here, we report the first exception to this rule. We cloned the CDase cDNA, consisting of 2142 nucleotides encoding 714 amino-acid residues, from the slime mould, Dictyostelium discoideum. The putative amino-acid sequence indicates 32-42% identity with various neutral CDases, but does not show any similarity to the acid and alkaline CDases, indicating the enzyme should be classified as a neutral CDase. However, overexpression of the cDNA in D. discoideum resulted in increased CDase activity at an acidic, but not a neutral pH range. Knockout of the gene in slime mould eliminated CDase activity at acidic pH. The recombinant enzyme expressed in the slime mould was purified and then characterized. Consequently, the purified CDase was found to exhibit the maximal activity at approx. pH 3.0. The singular pH dependency of slime mould CDase is not derived from the specific post-translational modification in the slime mould, because the enzyme showed an acidic pH optimum even when expressed in Chinese hamster ovary cells, whereas rat neutral-CDase exhibited a neutral pH optimum when expressed in slime mould. PMID:12943537

  1. Multi-scale interactions in Dictyostelium discoideum aggregation

    NASA Astrophysics Data System (ADS)

    Dixon, James A.; Kelty-Stephen, Damian G.

    2012-12-01

    Cellular aggregation is essential for a wide range of phenomena in developmental biology, and a crucial event in the life-cycle of Dictyostelium discoideum. The current manuscript presents an analysis of multi-scale interactions involved in D. discoideum aggregation and non-aggregation events. The multi-scale fractal dimensions of a sequence of microscope images were used to estimate changing structure at different spatial scales. Three regions showing aggregation and three showing non-aggregation were considered. The results showed that both aggregation and non-aggregation regions were strongly multi-fractal. Analyses of the over-time relationships among nine scales of the generalized dimension, D(q), were conducted using vector autoregression and vector error-correction models. Both types of regions showed evidence that across-scale interactions serve to maintain the equilibrium of the system. Aggregation and non-aggregation regions also showed different patterns of effects of individual scales on other scales. Specifically, aggregation regions showed greater effects of both the smallest and largest scales on the smaller scale structures. The results suggest that multi-scale interactions are responsible for maintaining and altering the cellular structures during aggregation.

  2. The genome of the social amoeba Dictyostelium discoideum

    PubMed Central

    Eichinger, L.; Pachebat, J.A.; Glöckner, G.; Rajandream, M.-A.; Sucgang, R.; Berriman, M.; Song, J.; Olsen, R.; Szafranski, K.; Xu, Q.; Tunggal, B.; Kummerfeld, S.; Madera, M.; Konfortov, B. A.; Rivero, F.; Bankier, A. T.; Lehmann, R.; Hamlin, N.; Davies, R.; Gaudet, P.; Fey, P.; Pilcher, K.; Chen, G.; Saunders, D.; Sodergren, E.; Davis, P.; Kerhornou, A.; Nie, X.; Hall, N.; Anjard, C.; Hemphill, L.; Bason, N.; Farbrother, P.; Desany, B.; Just, E.; Morio, T.; Rost, R.; Churcher, C.; Cooper, J.; Haydock, S.; van Driessche, N.; Cronin, A.; Goodhead, I.; Muzny, D.; Mourier, T.; Pain, A.; Lu, M.; Harper, D.; Lindsay, R.; Hauser, H.; James, K.; Quiles, M.; Babu, M. Madan; Saito, T.; Buchrieser, C.; Wardroper, A.; Felder, M.; Thangavelu, M.; Johnson, D.; Knights, A.; Loulseged, H.; Mungall, K.; Oliver, K.; Price, C.; Quail, M.A.; Urushihara, H.; Hernandez, J.; Rabbinowitsch, E.; Steffen, D.; Sanders, M.; Ma, J.; Kohara, Y.; Sharp, S.; Simmonds, M.; Spiegler, S.; Tivey, A.; Sugano, S.; White, B.; Walker, D.; Woodward, J.; Winckler, T.; Tanaka, Y.; Shaulsky, G.; Schleicher, M.; Weinstock, G.; Rosenthal, A.; Cox, E.C.; Chisholm, R. L.; Gibbs, R.; Loomis, W. F.; Platzer, M.; Kay, R. R.; Williams, J.; Dear, P. H.; Noegel, A. A.; Barrell, B.; Kuspa, A.

    2005-01-01

    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 encode ~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 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 appears to have retained more of the diversity of the ancestral genome than either of these two groups. PMID:15875012

  3. Systematic evaluation of buffer influences on the development of Dictyostelium discoideum.

    PubMed

    Márquez López, Johanna; Sulzmann, Anja; Thewes, Sascha

    2016-01-01

    Development and cell differentiation are key features of the social amoeba Dictyostelium discoideum. Already at early developmental stages, the gene expression profile changes in the amoebae to make the cells aggregation competent. In the laboratory, development starts when the cells are washed free of nutrients. For this purpose, various non-nutrient buffers are used in different laboratories. However, to date, it is not clear if different buffers have different influences on the development of D. discoideum. Therefore, we investigated systematically the influence of six widely used buffers on the development of D. discoideum. Investigation was done at the phenotypical, biochemical, and molecular level. The results show that some of the investigated buffers show clear differences in the phenotypical outcome of the developmental cycle, at a biochemical level as measured in the response to cAMP, and/or at a molecular level as measured in the expression of early developmental marker genes. According to our results buffer compositions should be considered carefully for all developmental experiments with D. discoideum, especially when gene expression will be investigated.

  4. Expression of the human muscarinic receptor gene m2 in Dictyostelium discoideum

    SciTech Connect

    Voith, G.; Dingermann, T.

    1995-11-01

    We have expressed a functional human muscarinic M2 receptor, under the control of the homologous discoidin I{gamma} promoter, in the cellular slime mold Dictyostelium discoideum. The use of a contact site A leader peptide ensured insertion of the newly synthesized receptor protein into the plasma membrane. Due to the characteristics of the discoidin I{gamma} promoter, the M2 receptor is expressed during late growth and early development. The heterologously expressed M2 receptors show binding characteristics similar to authentic receptors. Membranes as well as whole cells can be used in ligand binding assays. 36 refs., 4 figs.

  5. Chemotactic Blebbing in Dictyostelium Cells.

    PubMed

    Zatulovskiy, Evgeny; Kay, Robert R

    2016-01-01

    Many researchers use the social amoeba Dictyostelium discoideum as a model organism to study various aspects of the eukaryotic cell chemotaxis. Traditionally, Dictyostelium chemotaxis is considered to be driven mainly by branched F-actin polymerization. However, recently it has become evident that Dictyostelium, as well as many other eukaryotic cells, can also employ intracellular hydrostatic pressure to generate force for migration. This process results in the projection of hemispherical plasma membrane protrusions, called blebs, that can be controlled by chemotactic signaling.Here we describe two methods to study chemotactic blebbing in Dictyostelium cells and to analyze the intensity of the blebbing response in various strains and under different conditions. The first of these methods-the cyclic-AMP shock assay-allows one to quantify the global blebbing response of cells to a uniform chemoattractant stimulation. The second one-the under-agarose migration assay-induces directional blebbing in cells moving in a gradient of chemoattractant. In this assay, the cells can be switched from a predominantly F-actin-driven mode of motility to a bleb-driven chemotaxis, allowing one to compare the efficiency of both modes and explore the molecular machinery controlling chemotactic blebbing.

  6. Dictyostelium discoideum protein phosphatase-1 catalytic subunit exhibits distinct biochemical properties.

    PubMed Central

    Andrioli, Luiz P M; Zaini, Paulo A; Viviani, Wladia; Da Silva, Aline M

    2003-01-01

    Protein phosphatase-1 (PP1) is expressed ubiquitously and is involved in many eukaryotic cellular functions, although PP1 enzyme activity could not be detected in the social amoeba Dictyostelium discoideum cell extracts. In the present paper, we show that D. discoideum has a single copy gene that codes for the catalytic subunit of PP1 (DdPP1c). DdPP1c is expressed throughout the D. discoideum life cycle with constant levels of mRNA, and its protein and amino acid sequence show a mean identity of 80% with other PP1c enzymes. However, it has a distinctive difference: the substitution of a phenylalanine residue (Phe(269) in the DdPP1c) for a highly conserved cysteine residue (Cys(273) in rabbit PP1c) in a region that was shown to have a critical role in the interaction of rabbit PP1c with toxin inhibitors. Wild-type DdPP1c and an engineered mutant form in which Phe(269) was replaced by a cysteine residue were expressed in Escherichia coli. Both recombinant activities were similarly inhibited by okadaic acid, tautomycin and microcystin. However, the Phe(269)-->Cys mutation resulted in a large increase in enzyme activity towards phosphorylase a and a higher sensitivity to calyculin A. These results, together with the molecular modelling of DdPP1c structure, indicate that the Phe(269) residue, which occurs naturally in D. discoideum, confers distinct biochemical properties on this enzyme. PMID:12737629

  7. Deficiency of huntingtin has pleiotropic effects in the social amoeba Dictyostelium discoideum.

    PubMed

    Myre, Michael A; Lumsden, Amanda L; Thompson, Morgan N; Wasco, Wilma; MacDonald, Marcy E; Gusella, James F

    2011-04-01

    Huntingtin is a large HEAT repeat protein first identified in humans, where a polyglutamine tract expansion near the amino terminus causes a gain-of-function mechanism that leads to selective neuronal loss in Huntington's disease (HD). Genetic evidence in humans and knock-in mouse models suggests that this gain-of-function involves an increase or deregulation of some aspect of huntingtin's normal function(s), which remains poorly understood. As huntingtin shows evolutionary conservation, a powerful approach to discovering its normal biochemical role(s) is to study the effects caused by its deficiency in a model organism with a short life-cycle that comprises both cellular and multicellular developmental stages. To facilitate studies aimed at detailed knowledge of huntingtin's normal function(s), we generated a null mutant of hd, the HD ortholog in Dictyostelium discoideum. Dictyostelium cells lacking endogenous huntingtin were viable but during development did not exhibit the typical polarized morphology of Dictyostelium cells, streamed poorly to form aggregates by accretion rather than chemotaxis, showed disorganized F-actin staining, exhibited extreme sensitivity to hypoosmotic stress, and failed to form EDTA-resistant cell-cell contacts. Surprisingly, chemotactic streaming could be rescued in the presence of the bivalent cations Ca(2+) or Mg(2+) but not pulses of cAMP. Although hd(-) cells completed development, it was delayed and proceeded asynchronously, producing small fruiting bodies with round, defective spores that germinated spontaneously within a glassy sorus. When developed as chimeras with wild-type cells, hd(-) cells failed to populate the pre-spore region of the slug. In Dictyostelium, huntingtin deficiency is compatible with survival of the organism but renders cells sensitive to low osmolarity, which produces pleiotropic cell autonomous defects that affect cAMP signaling and as a consequence development. Thus, Dictyostelium provides a novel haploid

  8. A novel human receptor involved in bitter tastant detection identified using Dictyostelium discoideum.

    PubMed

    Robery, Steven; Tyson, Richard; Dinh, Christopher; Kuspa, Adam; Noegel, Angelika A; Bretschneider, Till; Andrews, Paul L R; Williams, Robin S B

    2013-12-01

    Detection of substances tasting bitter to humans occurs in diverse organisms including the social amoeba Dictyostelium discoideum. To establish a molecular mechanism for bitter tastant detection in Dictyostelium, we screened a mutant library for resistance to a commonly used bitter standard, phenylthiourea. This approach identified a G-protein-coupled receptor mutant, grlJ(-), which showed a significantly increased tolerance to phenylthiourea in growth, survival and movement. This mutant was not resistant to a structurally dissimilar potent bitter tastant, denatonium benzoate, suggesting it is not a target for at least one other bitter tastant. Analysis of the cell-signalling pathway involved in the detection of phenylthiourea showed dependence upon heterotrimeric G protein and phosphatidylinositol 3-kinase activity, suggesting that this signalling pathway is responsible for the cellular effects of phenylthiourea. This is further supported by a phenylthiourea-dependent block in the transient cAMP-induced production of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) in wild-type but not grlJ(-) cells. Finally, we have identified an uncharacterized human protein γ-aminobutyric acid (GABA) type B receptor subunit 1 isoform with weak homology to GrlJ that restored grlJ(-) sensitivity to phenylthiourea in cell movement and PIP3 regulation. Our results thus identify a novel pathway for the detection of the standard bitter tastant phenylthiourea in Dictyostelium and implicate a poorly characterized human protein in phenylthiourea-dependent cell responses.

  9. NaCS-PDMDAAC immobilized cultivation of recombinant Dictyostelium discoideum for soluble human Fas ligand production.

    PubMed

    Zheng, Chao; Zeng, Xianhai; Danquah, Michael K; Lu, Yinghua

    2015-01-01

    Dictyostelium discoideum is a promising eukaryotic host for the expression of heterologous proteins requiring post-translational modifications. However, the dilute nature of D. discoideum cell culture limits applications for high value proteins production. D. discoideum cells, entrapped in sodium cellulose sulfate/poly-dimethyl-diallyl-ammonium chloride (NaCS-PDMDAAC) capsules were used for biosynthesis of the heterologous protein, soluble human Fas ligand (hFasL). Semi-continuous cultivations with capsules recycling were carried out in shake flasks. Also, a scaled-up cultivation of immobilized D. discoideum for hFasL production in a customized vitreous airlift bioreactor was conducted. The results show that NaCS-PDMDAAC capsules have desirable biophysical properties including biocompatibility with the D. discoideum cells and good mechanical stability throughout the duration of cultivation. A maximum cell density of 2.02 × 10(7) cells mL(-1) (equivalent to a maximum cell density of 2.22 × 10(8) cells mL(-1) in capsules) and a hFasL concentration of 130.40 μg L(-1) (equivalent to a hFasL concentration of 1434.40 μg L(-1) in capsules) were obtained in shake flask cultivation with capsules recycling. Also, a maximum cell density of 1.72 × 10(7) cells mL(-1) (equivalent to a maximum cell density of 1.89 × 10(8) cells mL(-1) in capsules) and a hFasL concentration of 106.10 μg L(-1) (equivalent to a hFasL concentration of 1167.10 μg L(-1) in capsules) were obtained after ∼170 h cultivation in the airlift bioreactor (with a working volume of 200 mL in a 315 mL bioreactor). As the article presents a premier work in the application of NaCS-PDMDAAC immobilized D. discoideum cells for the production of hFasL, more work is required to further optimize the system to generate higher cell densities and hFasL titers for large-scale applications.

  10. A continuum analysis of the chemotactic signal seen by Dictyostelium discoideum.

    PubMed

    Dallon, J C; Othmer, H G

    1998-10-21

    We developed a mathematical model of cell-to-cell-signalling in Dictyostelium discoideum that predicts the cAMP signal seen by individual cells in early aggregation. The model employs two cells on a plane and is designed to predict the space-time characteristics of both the extracellular cAMP signal seen by one cell when a nearby cell relays, and the intracellular cAMP response produced by the stimulus in the receiving cell. The effect of membrane bound phosphodiesterase is studied and it is shown that cells can orient effectively even in its absence. Our results give a detailed picture of how the spatio-temporal characteristics of the extracellular signal can be transduced into a time- and space-dependent intracellular gradient, and they suggest a plausible mechanism for orientation in a natural chemotactic wave.

  11. Dictyostelium discoideum Nucleoside Diphosphate Kinase C Plays a Negative Regulatory Role in Phagocytosis, Macropinocytosis and Exocytosis

    PubMed Central

    Annesley, Sarah J.; Bago, Ruzica; Bosnar, Maja Herak; Filic, Vedrana; Marinović, Maja; Weber, Igor; Mehta, Anil; Fisher, Paul R.

    2011-01-01

    Nucleoside diphosphate kinases (NDPKs) are ubiquitous phosphotransfer enzymes responsible for producing most of the nucleoside triphosphates except for ATP. This role is important for the synthesis of nucleic acids and proteins and the metabolism of sugars and lipids. Apart from this housekeeping role NDPKs have been shown to have many regulatory functions in diverse cellular processes including proliferation and endocytosis. Although the protein has been shown to have a positive regulatory role in clathrin- and dynamin-mediated micropinocytosis, its roles in macropinocytosis and phagocytosis have not been studied. The additional non-housekeeping roles of NDPK are often independent of enzyme activity but dependent on the expression level of the protein. In this study we altered the expression level of NDPK in the model eukaryotic organism Dictyostelium discoideum through antisense inhibition and overexpression. We demonstrate that NDPK levels affect growth, endocytosis and exocytosis. In particular we find that Dictyostelium NDPK negatively regulates endocytosis in contrast to the positive regulatory role identified in higher eukaryotes. This can be explained by the differences in types of endocytosis that have been studied in the different systems - phagocytosis and macropinocytosis in Dictyostelium compared with micropinocytosis in mammalian cells. This is the first report of a role for NDPK in regulating macropinocytosis and phagocytosis, the former being the major fluid phase uptake mechanism for macrophages, dendritic cells and other (non dendritic) cells exposed to growth factors. PMID:21991393

  12. The thyroxine inactivating gene, type III deiodinase, suppresses multiple signaling centers in Dictyostelium discoideum.

    PubMed

    Singh, Shashi Prakash; Dhakshinamoorthy, Ranjani; Jaiswal, Pundrik; Schmidt, Stefanie; Thewes, Sascha; Baskar, Ramamurthy

    2014-12-15

    Thyroxine deiodinases, the enzymes that regulate thyroxine metabolism, are essential for vertebrate growth and development. In the genome of Dictyostelium discoideum, a single intronless gene (dio3) encoding type III thyroxine 5' deiodinase is present. The amino acid sequence of D. discoideum Dio3 shares 37% identity with human T4 deiodinase and is a member of the thioredoxin reductase superfamily. dio3 is expressed throughout growth and development and by generating a knockout of dio3, we have examined the role of thyroxine 5' deiodinase in D. discoideum. dio3(-) had multiple defects that affected growth, timing of development, aggregate size, cell streaming, and cell-type differentiation. A prominent phenotype of dio3(-) was the breaking of late aggregates into small signaling centers, each forming a fruiting body of its own. cAMP levels, its relay, photo- and chemo-taxis were also defective in dio3(-). Quantitative RT-PCR analyses suggested that expression levels of genes encoding adenylyl cyclase A (acaA), cAMP-receptor A (carA) and cAMP-phosphodiesterases were reduced. There was a significant reduction in the expression of CadA and CsaA, which are involved in cell-cell adhesion. The dio3(-) slugs had prestalk identity, with pronounced prestalk marker ecmA expression. Thus, Dio3 seems to have roles in mediating cAMP synthesis/relay, cell-cell adhesion and slug patterning. The phenotype of dio3(-) suggests that Dio3 may prevent the formation of multiple signaling centers during D. discoideum development. This is the first report of a gene involved in thyroxine metabolism that is also involved in growth and development in a lower eukaryote.

  13. Assets of the non-pathogenic microorganism Dictyostelium discoideum as a model for the study of eukaryotic extracellular vesicles.

    PubMed

    Tatischeff, Irène

    2013-01-01

    Dictyostelium discoideum microvesicles have recently been presented as a valuable model for eukaryotic extracellular vesicles. Here, the advantages of D. discoideum for unraveling important biological functions of extracellular vesicles in general are detailed. D. discoideum, a non-pathogenic eukaryotic microorganism, belongs to a billion-year-old Amoeboza lineage, which diverged from the animal-fungal lineage after the plant animal-split. During growth and early starvation-induced development, it presents analogies with lymphocytes and macrophages with regard to motility and phagocytosis capability, respectively. Its 6-chromosome genome codes for about 12,500 genes, some showing analogies with human genes. The presence of extracellular vesicles during cell growth has been evidenced as a detoxification mechanism of various structurally unrelated drugs. Controls led to the discovery of constitutive extracellular vesicle secretion in this microorganism, which was an important point. It means that the secretion of extracellular vesicles occurs, in the absence of any drug, during both cell growth and early development. This constitutive secretion of D. discoideum cells is very likely to play a role in intercellular communication. The detoxifying secreted vesicles, which can transport drugs outside the cells, can also act as "Trojan horses", capable of transferring these drugs not only into naïve D. discoideum cells, but into human cells as well. Therefore, these extracellular vesicles were proposed as a new biological drug delivery tool. Moreover, Dictyostelium, chosen by the NIH (USA) as a new model organism for biomedical research, has already been used for studying some human diseases. These cells, which are much easier to manipulate than human cells, can be easily designed in simple conditioned medium experiments. Owing to the increasing consensus that extracellular vesicles are probably important mediators of intercellular communication, D. discoideum is here

  14. Assets of the non-pathogenic microorganism Dictyostelium discoideum as a model for the study of eukaryotic extracellular vesicles.

    PubMed

    Tatischeff, Irène

    2013-03-04

    Dictyostelium discoideum microvesicles have recently been presented as a valuable model for eukaryotic extracellular vesicles. Here, the advantages of D. discoideum for unraveling important biological functions of extracellular vesicles in general are detailed. D. discoideum, a non-pathogenic eukaryotic microorganism, belongs to a billion-year-old Amoeboza lineage, which diverged from the animal-fungal lineage after the plant animal-split. During growth and early starvation-induced development, it presents analogies with lymphocytes and macrophages with regard to motility and phagocytosis capability, respectively. Its 6-chromosome genome codes for about 12,500 genes, some showing analogies with human genes. The presence of extracellular vesicles during cell growth has been evidenced as a detoxification mechanism of various structurally unrelated drugs. Controls led to the discovery of constitutive extracellular vesicle secretion in this microorganism, which was an important point. It means that the secretion of extracellular vesicles occurs, in the absence of any drug, during both cell growth and early development. This constitutive secretion of D. discoideum cells is very likely to play a role in intercellular communication. The detoxifying secreted vesicles, which can transport drugs outside the cells, can also act as "Trojan horses", capable of transferring these drugs not only into naïve D. discoideum cells, but into human cells as well. Therefore, these extracellular vesicles were proposed as a new biological drug delivery tool. Moreover, Dictyostelium, chosen by the NIH (USA) as a new model organism for biomedical research, has already been used for studying some human diseases. These cells, which are much easier to manipulate than human cells, can be easily designed in simple conditioned medium experiments. Owing to the increasing consensus that extracellular vesicles are probably important mediators of intercellular communication, D. discoideum is here

  15. Self-organized, near-critical behavior during aggregation in Dictyostelium discoideum

    NASA Astrophysics Data System (ADS)

    de Palo, Giovanna; Yi, Darvin; Gregor, Thomas; Endres, Robert

    During starvation, the social amoeba Dictyostelium discoideum aggregates artfully via pattern formation into a multicellular slug and finally spores. The aggregation process is mediated by the secretion and sensing of cyclic adenosine monophosphate, leading to the synchronized movement of cells. The whole process is a remarkable example of collective behavior, spontaneously emerging from single-cell chemotaxis. Despite this phenomenon being broadly studied, a precise characterization of the transition from single cells to multicellularity has been elusive. Here, using fluorescence imaging data of thousands of cells, we investigate the role of cell shape in aggregation, demonstrating remarkable transitions in cell behavior. To better understand their functional role, we analyze cell-cell correlations and provide evidence for self-organization at the onset of aggregation (as opposed to leader cells), with features of criticality in this finite system. To capture the mechanism of self-organization, we extend a detailed single-cell model of D.discoideum chemotaxis by adding cell-cell communication. We then use these results to extract a minimal set of rules leading to aggregation in the population model. If universal, similar rules may explain other types of collective cell behavior.

  16. The Social Amoeba Dictyostelium discoideum Is Highly Resistant to Polyglutamine Aggregation.

    PubMed

    Santarriaga, Stephanie; Petersen, Amber; Ndukwe, Kelechi; Brandt, Anthony; Gerges, Nashaat; Bruns Scaglione, Jamie; Scaglione, Kenneth Matthew

    2015-10-16

    The expression, misfolding, and aggregation of long repetitive amino acid tracts are a major contributing factor in a number of neurodegenerative diseases, including C9ORF72 amyotrophic lateral sclerosis/frontotemporal dementia, fragile X tremor ataxia syndrome, myotonic dystrophy type 1, spinocerebellar ataxia type 8, and the nine polyglutamine diseases. Protein aggregation is a hallmark of each of these diseases. In model organisms, including yeast, worms, flies, mice, rats, and human cells, expression of proteins with the long repetitive amino acid tracts associated with these diseases recapitulates the protein aggregation that occurs in human disease. Here we show that the model organism Dictyostelium discoideum has evolved to normally encode long polyglutamine tracts and express these proteins in a soluble form. We also show that Dictyostelium has the capacity to suppress aggregation of a polyglutamine-expanded Huntingtin construct that aggregates in other model organisms tested. Together, these data identify Dictyostelium as a novel model organism with the capacity to suppress aggregation of proteins with long polyglutamine tracts.

  17. A non-mitotic CENP-E homolog in Dictyostelium discoideum with slow motor activity.

    PubMed

    Kösem, Süleyman; Ökten, Zeynep; Ho, Thi-Hieu; Trommler, Gudrun; Koonce, Michael P; Samereier, Matthias; Müller-Taubenberger, Annette

    2013-02-15

    Kinesins are ATP-dependent molecular motors that mediate unidirectional intracellular transport along microtubules. Dictyostelium discoideum has 13 different kinesin isoforms including two members of the kinesin-7 family, Kif4 and Kif11. While Kif4 is structurally and functionally related to centromere-associated CENP-E proteins involved in the transport of chromosomes to the poles during mitosis, the function of the unusually short CENP-E variant Kif11 is unclear. Here we show that orthologs of short CENP-E variants are present in plants and fungi, and analyze functional properties of the Dictyostelium CENP-E version, Kif11. Gene knockout mutants reveal that Kif11 is not required for mitosis or development. Imaging of GFP-labeled Kif11 expressing Dictyostelium cells indicates that Kif11 is a plus-end directed motor that accumulates at microtubule plus ends. By multiple motor gliding assays, we show that Kif11 moves with an average velocity of 38nm/s, thus defining Kif11 as a very slow motor. The activity of the Kif11 motor appears to be modulated via interactions with the non-catalytic tail region. Our work highlights a subclass of kinesin-7-like motors that function outside of a role in mitosis.

  18. Effects of medicinal compounds on the differentiation of the eukaryotic microorganism dictyostelium discoideum: can this model be used as a screening test for reproductive toxicity in humans?

    PubMed

    Dannat, K; Tillner, J; Winckler, T; Weiss, M; Eger, K; Dingermann, T

    2003-03-01

    Dictyostelium discoideum is a single-cell, eukaryotic microorganism that can undergo multicellular development in order to produce dormant spores. We investigated the capacity of D. discoideum to be used as a rapid screening system for potential developmental toxicity of compounds under development as pharmaceuticals. We used a set of four transgenic D. discoideum strains that expressed a reporter gene under the control of promoters that are active at certain time periods and in distinct cell types during D. discoideum development. We found that teratogens such as valproic acid, tretinoin, or thalidomide interfered to various extents with D. discoideum development, and had different effects on prestalk and prespore cell-specific reporter gene expression. Phenytoin was inactive in this assay, which may point to limitations in metabolization of the compound in Dictyostelium required to exert developmental toxicity. D. discoideum cell culture is cheap and easy to handle compared to mammalian cell cultures or animal teratogenicity models. Although the Dictyostelium-based assay described in this report may not securely predict the teratogenic potential of these drugs in humans, this organism may be qualified for rapid large-scale screenings of synthetic compounds under development as new pharmaceuticals for their potential to interfere with developmental processes and thus help to reduce the amount of teratogenicity tests in animal models.

  19. A retinoblastoma orthologue is required for the sensing of a chalone in Dictyostelium discoideum.

    PubMed

    Bakthavatsalam, Deenadayalan; White, Michael J V; Herlihy, Sarah E; Phillips, Jonathan E; Gomer, Richard H

    2014-03-01

    Retinoblastoma-like proteins regulate cell differentiation and inhibit cell proliferation. The Dictyostelium discoideum retinoblastoma orthologue RblA affects the differentiation of cells during multicellular development, but it is unclear whether RblA has a significant effect on Dictyostelium cell proliferation, which is inhibited by the secreted proteins AprA and CfaD. We found that rblA⁻ cells in shaking culture proliferate to a higher density, die faster after reaching stationary density, and, after starvation, have a lower spore viability than wild-type cells, possibly because in shaking culture, rblA⁻ cells have both increased cytokinesis and lower extracellular accumulation of CfaD. However, rblA⁻ cells have abnormally slow proliferation on bacterial lawns. Recombinant AprA inhibits the proliferation of wild-type cells but not that of rblA⁻ cells, whereas CfaD inhibits the proliferation of both wild-type cells and rblA⁻ cells. Similar to aprA⁻ cells, rblA⁻ cells have a normal mass and protein accumulation rate on a per-nucleus basis, indicating that RblA affects cell proliferation but not cell growth. AprA also functions as a chemorepellent, and RblA is required for proper AprA chemorepellent activity despite the fact that RblA does not affect cell speed. Together, our data indicate that an autocrine proliferation-inhibiting factor acts through RblA to regulate cell density in Dictyostelium, suggesting that such factors may signal through retinoblastoma-like proteins to control the sizes of structures such as developing organs or tumors.

  20. Nucleotide sequence of alkyl-dihydroxyacetonephosphate synthase cDNA from Dictyostelium discoideum.

    PubMed

    de Vet, E C; van den Bosch, H

    1998-11-27

    The nucleotide sequence is reported of alkyl-dihydroxyacetonephosphate synthase cDNA from the cellular slime mold Dictyostelium discoideum. The open reading frame encodes a protein of 611 amino acids which shows a 33% amino acid identity to the human enzyme. This D. discoideum homolog carries a variant of the peroxisomal targeting signal type 1 at its C-terminus (PKL). Expression of the cDNA in Escherichia coli yielded an enzymatically active protein.

  1. Functional analysis of a novel gene, DD3-3, from Dictyostelium discoideum

    SciTech Connect

    Sakuragi, N.; Ogasawara, N.; Tanesaka, E.; Yoshida, M. . E-mail: yoshida_m@nara.kindai.ac.jp

    2005-06-17

    A novel gene, DD3-3, from Dictyostelium discoideum has been isolated by an mRNA differential display between a wild-type strain AX2 and a mutant HG794 which is defective in O-glycosylation. Functional analysis of the novel gene, DD3-3, was conducted by preparing a knockout mutant, DD3-3KO, and a GST:DD3-3 fusion protein. The mutant DD3-3KO cells were allowed to develop about 1.5 h earlier than the wild-type strain AX2 cells. Northern blotting analysis of the knockout mutant cells showed a remarkable downregulation of Reg A, cAMP-dependent phosphodiesterase, and overexpression of protein tyrosine kinase (PTK) during early development and its shutdown during late development. The relationship between O-glycosylation and phosphorylation involving Reg A gene is discussed.

  2. Origin and Evolution of Circular Waves and Spirals in Dictyostelium discoideum Territories

    NASA Astrophysics Data System (ADS)

    Palsson, Eirikur; Cox, Edward C.

    1996-02-01

    Randomly distributed Dictyostelium discoideum cells form cooperative territories by signaling to each other with cAMP. Cells initiate the process by sending out pulsatile signals, which propagate as waves. With time, circular and spiral patterns form. We show that by adding spatial and temporal noise to the levels of an important regulator of external cAMP levels, the cAMP phosphodiesterase inhibitor, we can explain the natural progression of the system from randomly firing cells to circular waves whose symmetries break to form double- and single- or multi-armed spirals. When phosphodiesterase inhibitor is increased with time, mimicking experimental data, the wavelength of the spirals shortens, and a proportion of them evolve into pairs of connected spirals. We compare these results to recent experiments, finding that the temporal and spatial correspondence between experiment and model is very close.

  3. Cyclic AMP stabilizes a class of developmentally regulated Dictyostelium discoideum mRNAs.

    PubMed

    Mangiarotti, G; Ceccarelli, A; Lodish, H F

    The stability of mRNA is an important facet of the regulation of protein synthesis. In mammalian cells most mRNAs have long half-lives (5-15 hours) but a substantial fraction are much less stable. There are few examples where the stability of a particular mRNA or class of mRNAs is specifically affected by environmental or developmental stimuli. Certain hormones cause specific stabilization of mRNAs species and preferential mRNA stability is important in the accumulation of globin and myosin mRNAs during the terminal stages of erythropoesis or myogenesis, respectively. Disaggregation of Dictyostelium discoideum aggregates induces the specific destabilization of a large class of developmentally regulated mRNAs; thus, this system is an excellent one in which to determine how such controls are effected. Here we show that addition of cyclic AMP to disaggregated cells specifically prevents the destabilization of these mRNAs.

  4. An ancestral non-proteolytic role for presenilin proteins in multicellular development of the social amoeba Dictyostelium discoideum.

    PubMed

    Ludtmann, Marthe H R; Otto, Grant P; Schilde, Christina; Chen, Zhi-Hui; Allan, Claire Y; Brace, Selina; Beesley, Philip W; Kimmel, Alan R; Fisher, Paul; Killick, Richard; Williams, Robin S B

    2014-04-01

    Mutations in either of two presenilin genes can cause familial Alzheimer's disease. Presenilins have both proteolysis-dependent functions, as components of the γ-secretase complex, and proteolysis-independent functions in signalling. In this study, we investigate a conserved function of human presenilins in the development of the simple model organism Dictyostelium discoideum. We show that the block in Dictyostelium development caused by the ablation of both Dictyostelium presenilins is rescued by the expression of human presenilin 1, restoring the terminal differentiation of multiple cell types. This developmental role is independent of proteolytic activity, because the mutation of both catalytic aspartates does not affect presenilin ability to rescue development, and the ablation of nicastrin, a γ-secretase component that is crucial for proteolytic activity, does not block development. The role of presenilins during Dictyostelium development is therefore independent of their proteolytic activity. However, presenilin loss in Dictyostelium results in elevated cyclic AMP (cAMP) levels and enhanced stimulation-induced calcium release, suggesting that presenilins regulate these intracellular signalling pathways. Our data suggest that presenilin proteins perform an ancient non-proteolytic role in regulating intracellular signalling and development, and that Dictyostelium is a useful model for analysing human presenilin function.

  5. Some repair-deficient mutants of Dictyostelium discoideum display enhanced susceptibilities to bleomycin.

    PubMed Central

    Deering, R A; Guyer, R B; Stevens, L; Watson-Thais, T E

    1996-01-01

    Dictyostelium discoideum, a soil eukaryote, is highly resistant to DNA-damaging agents; repair mutants are more susceptible. Susceptibility to bleomycin, produced by Streptomyces verticillus, is greater for mutants which are susceptible to other agents than for resistant strains. The high potential for DNA repair may result from the need to cope with chemicals produced by other soil microorganisms. PMID:8834899

  6. Flow-driven two-dimensional waves in colonies of Dictyostelium discoideum

    NASA Astrophysics Data System (ADS)

    Gholami, A.; Zykov, V.; Steinbock, O.; Bodenschatz, E.

    2015-09-01

    Dictyostelium discoideum (D.d.) is a valuable model organism to study self-organization and pattern formation in biology. Recently we reported flow-driven waves in experiments with uniformly distributed populations of signaling amobae, D.d., and carried out a theoretical study in a one-dimensional model. In this work, we perform two-dimensional numerical simulations using the well-known Martiel-Golbeter model to study the effect of the flow profile and intrinsic noise on the flow-driven waves. We show that, in the presence of flow, a persistence noise due to spontaneous cell firing events can lead to sustained structures that fill the whole length of the system. We also show that external periodic stimuli of cyclic adenosine monophosphate can induce 1:1 and 2:1 entrainments which are in agreement with our experimental observations.

  7. Specific mRNA destabilization in Dictyostelium discoideum requires RNA synthesis.

    PubMed Central

    Amara, J F; Lodish, H F

    1987-01-01

    We tested the effects of inhibitors of protein and RNA synthesis on the disaggregation-mediated destabilization of prespore mRNAs in Dictyostelium discoideum. Incubating disaggregated cells with daunomycin to inhibit RNA synthesis prevented the loss of prespore mRNAs, whereas the inhibitor decreased or did not affect levels of the common mRNAs CZ22 and actin. Protein synthesis inhibitors varied in their effects. Cycloheximide, which inhibited protein synthesis almost completely, prevented the loss of the prespore mRNAs, but puromycin, which inhibited protein synthesis less well, did not. These results indicate that the process of specific mRNA destabilization requires the synthesis of RNA and possibly of protein. Images PMID:3437899

  8. A RabGAP Regulates Life-Cycle Duration via Trimeric G-protein Cascades in Dictyostelium discoideum

    PubMed Central

    Kuwayama, Hidekazu; Miyanaga, Yukihiro; Urushihara, Hideko; Ueda, Masahiro

    2013-01-01

    Background The life-cycle of cellular slime molds comprises chronobiologically regulated processes. During the growth phase, the amoeboid cells proliferate at a definite rate. Upon starvation, they synthesize cAMP as both first and second messengers in signalling pathways and form aggregates, migrating slugs, and fruiting bodies, consisting of spores and stalk cells, within 24 h. In Dictyostelium discoideum, because most growth-specific events cease during development, proliferative and heterochronic mutations are not considered to be interrelated and no genetic factor governing the entire life-cycle duration has ever been identified. Methodology/Principal Findings Using yeast 2-hybrid library screening, we isolated a Dictyostelium discoideum RabGAP, Dd Rbg-3, as a candidate molecule by which the Dictyostelium Gα2 subunit directs its effects. Rab GTPase-activating protein, RabGAP, acts as a negative regulator of Rab small GTPases, which orchestrate the intracellular membrane trafficking involved in cell proliferation. Deletion mutants of Dd rbg-3 exhibited an increased growth rate and a shortened developmental period, while an overexpression mutant demonstrated the opposite effects. We also show that Dd Rbg-3 interacts with 2 Gα subunits in an activity-dependent manner in vitro. Furthermore, both human and Caenorhabditis elegans rbg-3 homologs complemented the Dd rbg-3–deletion phenotype in D. discoideum, indicating that similar pathways may be generally conserved in multicellular organisms. Conclusions/Significance Our findings suggest that Dd Rbg-3 acts as a key element regulating the duration of D. discoideum life-span potentially via trimeric G-protein cascades. PMID:24349132

  9. Nucleocytoplasmic protein translocation during mitosis in the social amoebozoan Dictyostelium discoideum.

    PubMed

    O'Day, Danton H; Budniak, Aldona

    2015-02-01

    Mitosis is a fundamental and essential life process. It underlies the duplication and survival of all cells and, as a result, all eukaryotic organisms. Since uncontrolled mitosis is a dreaded component of many cancers, a full understanding of the process is critical. Evolution has led to the existence of three types of mitosis: closed, open, and semi-open. The significance of these different mitotic species, how they can lead to a full understanding of the critical events that underlie the asexual duplication of all cells, and how they may generate new insights into controlling unregulated cell division remains to be determined. The eukaryotic microbe Dictyostelium discoideum has proved to be a valuable biomedical model organism. While it appears to utilize closed mitosis, a review of the literature suggests that it possesses a form of mitosis that lies in the middle between truly open and fully closed mitosis-it utilizes a form of semi-open mitosis. Here, the nucleocytoplasmic translocation patterns of the proteins that have been studied during mitosis in the social amoebozoan D. discoideum are detailed followed by a discussion of how some of them provide support for the hypothesis of semi-open mitosis.

  10. Selection and analysis of cloned developmentally-regulated Dictyostelium discoideum genes by hybridization-competition.

    PubMed Central

    Mangiarotti, G; Chung, S; Zuker, C; Lodish, H F

    1981-01-01

    We describe a new technique for selection of cloned gene segments which are expressed preferentially at one developmental stage but at a relatively low level. A nitrocellulose filter replica of plaques of lambda phage which contain approximately 8 KB inserts of genomic DNA is prepared; it is hybridized with a small amount of [32p] labeled mRNA prepared from one developmental stage, in the presence of a several-hundred fold excess of competitor RNA from a different stage. We show that clones of Dictyostelium nuclear DNA which form hybrids under these conditions indeed encode developmentally regulated mRNAs. Our previous analysis of Dictyostelium discoideum differentiation indicated that transcripts from about 12% of the genome appear in mRNA at one defined stage of differentiation - the formation of cell-cell aggregates. A number of our new clones are novel, in that they encode multiple discrete mRNA species all of which accumulate only at the cell aggregate stages; others encode one or more mRNAs which appear at the tight aggregate stage and also one or more which are present throughout differentiation. These latter clones, in particular, would be difficult to identify using other selection techniques. Images PMID:7232208

  11. Dictyostelium discoideum Ax2 as an Assay System for Screening of Pharmacological Chaperones for Phenylketonuria Mutations.

    PubMed

    Kim, Yu-Min; Yang, Yun Gyeong; Kim, Hye-Lim; Park, Young Shik

    2015-06-01

    In this study, we developed an assay system for missense mutations in human phenylalanine hydroxylases (hPAHs). To demonstrate the reliability of the system, eight mutant proteins (F39L, K42I, L48S, I65T, R252Q, L255V, S349L, and R408W) were expressed in a mutant strain (pah(-)) of Dictyostelium discoideum Ax2 disrupted in the indigenous gene encoding PAH. The transformed pah- cells grown in FM minimal medium were measured for growth rate and PAH activity to reveal a positive correlation between them. The protein level of hPAH was also determined by western blotting to show the impact of each mutation on protein stability and catalytic activity. The result was highly compatible with the previous ones obtained from other expression systems, suggesting that Dictyostelium is a dependable alternative to other expression systems. Furthermore, we found that both the protein level and activity of S349L and R408W, which were impaired severely in protein stability, were rescued in HL5 nutrient medium. Although the responsible component(s) remains unidentified, this unexpected finding showed an important advantage of our expression system for studying unstable proteins. As an economic and stable cell-based expression system, our development will contribute to mass-screening of pharmacological chaperones for missense PAH mutations as well as to the in-depth characterization of individual mutations.

  12. Caspase-like proteins: Acanthamoeba castellanii metacaspase and Dictyostelium discoideum paracaspase, what are their functions?

    PubMed

    Saheb, Entsar; Trzyna, Wendy; Bush, John

    2014-12-01

    Caspases are cysteine proteases that are important regulators of programmed cell death in animals. Two novel relatives to members of the caspase families metacaspases and paracaspase have been discovered. Metacaspase type-1 was identified in Acanthamoeba castellanii, an opportunistic protozoan parasite that causes severe diseases in humans. Paracaspase was found in the non-pathogenic protozoan Dictyostelium discoideum. Since their discovery in Acanthamoeba and Dictyostelium, metacaspases and paracaspases have remained poorly characterized. At present we do not have sufficient data about the molecular function of these caspase-like proteins or their role, if any, in programmed cell death. How these caspase proteins function at the molecular level is an important area of study that will provide insight into their potential for treatment therapies against Acanthamoeba infection and other similar parasitic protozoan. Additionally, finding the molecular functions of these caspase-like proteins will provide information concerning their role in more complex organisms.The aim of this article was to review recent discoveries about metacaspases and paracaspases as regulators of apoptotic and non-apoptotic processes.

  13. Vmp1 regulates PtdIns3P signaling during autophagosome formation in Dictyostelium discoideum.

    PubMed

    Calvo-Garrido, Javier; King, Jason S; Muñoz-Braceras, Sandra; Escalante, Ricardo

    2014-11-01

    Generation and turnover of phosphatidylinositol 3-phosphate (PtdIns3P) signaling is essential for autophagosome formation and other membrane traffic processes. In both Dictyostelium discoideum and mammalian cells, autophagosomes are formed from specialized regions of the endoplasmic reticulum (ER), called omegasomes, which are enriched in the signaling lipid PtdIns3P. Vacuole membrane protein 1 (Vmp1) is a multispanning membrane protein localized at the ER that is required for autophagosome formation. There are conflicting reports in the literature as to whether Vmp1 is strictly required or not for autophagy-related PtdIns3P signaling and its hierarchical relationship with Atg1 and PI3K. We have now addressed these questions in the Dictyostelium model. We show that Dictyostelium cells lacking Vmp1 have elevated and aberrant PtdIns3P signaling on the ER, resulting in an increased and persistent recruitment of Atg18 and other autophagic proteins. This indicates that Vmp1 is not strictly essential for the generation of PtdIns3P signaling but rather suggests a role in the correct turnover or modulation of this signaling. Of interest, these PtdIns3P-enriched regions of the ER surround ubiquitinated protein aggregates but are unable to form functional autophagosomes. vmp1 null cells also have additional defects in macropinocytosis and growth, which are not shared by other autophagy mutants. Remarkably, we show that these defects and also the aberrant PtdIns3P distribution are largely suppressed by the concomitant loss of Atg1, indicating that aberrant autophagic signaling on the ER inhibits macropinocytosis. These results suggest that Atg1 functions upstream of Vmp1 in this signaling pathway and demonstrates a previously unappreciated link between abnormal autophagy signaling and macropinocytosis.

  14. Mechanism of oligomerisation of cyclase-associated protein from Dictyostelium discoideum in solution.

    PubMed

    Yusof, Adlina Mohd; Jaenicke, Elmar; Pedersen, Jan Skov; Noegel, Angelika A; Schleicher, Michael; Hofmann, Andreas

    2006-10-06

    Cyclase-associated protein (CAP) is a highly conserved modular protein implicated in the regulation of actin filament dynamics and a variety of developmental and morphological processes. The protein exists as a high molecular weight complex in cell extracts and purified protein possesses a high tendency to aggregate, a major obstacle for crystallisation. Using a mutagenesis approach, we show that two structural features underlie the mechanism of oligomerisation in Dictyostelium discoideum CAP. Positively charged clusters on the surface of the N-terminal helix-barrel domain are involved in inter-molecular interactions with the N or C-terminal domains. Abolishing these interactions mainly renders dimers due to a domain swap feature in the extreme C-terminal region of the protein that was previously described. Based on earlier studies with yeast CAP, we also generated constructs with mutations in the extreme N-terminal region of Dictyostelium CAP that did not show significantly altered oligomerisation behaviour. Constructs with mutations in the earlier identified protein-protein interaction interface on the N-terminal domain of CAP could not be expressed as soluble protein. Assessment of the soluble proteins indicates that the mutations did not affect their overall fold. Further studies point to the correlation between stability of full-length CAP with its multimerisation behaviour, where oligomer formation leads to a more stable protein.

  15. Primary structure and regulation of vegetative specific genes of Dictyostelium discoideum.

    PubMed Central

    Singleton, C K; Manning, S S; Ken, R

    1989-01-01

    We have examined the expression and structure of several genes belonging to two classes of vegetative specific genes of the simple eukaryote, Dictyostelium discoideum. In amebae grown on bacteria, deactivation of all vegetative specific genes occurred at the onset of development and very little mRNA exists by 8 to 10 hours. In contrast, when cells were grown in axenic broth, the mRNA levels remained constant until a dramatic drop occurred around 10 to 12 hours. Thus, regulation of both classes of genes during the first several hours of development is dependent upon the prior growth conditions. Analysis of genomic clones has resulted in the identification of two V genes, V1 and V18, as ribosomal protein genes. Several other V genes were not found to be ribosomal protein genes, suggesting that in Dictyostelium non-ribosomal protein genes may be coordinately regulated with the ribosomal protein genes. Finally, using deletion analysis we show that the promoters of two of the V genes are composed of a constitutive positive element(s) located upstream of sequences involved in the regulated expression of these genes and within the first 545 upstream bp for V18 and 850 bp for V14. The regions involved in regulated expression were localized between -7 and -222 for V18 and -70 and -368 for V14. The sequences conferring protein synthesis sensitivity were shown to reside between -502 and -61 of the H4 promoter. Images PMID:2602140

  16. Novel zinc protease gene isolated from Dictyostelium discoideum is structurally related to mammalian leukotriene A4 hydrolase.

    PubMed

    Fan, D; Hou, L S

    2015-12-09

    The allantoicase (allC) gene of Dictyostelium discoideum allC RNAi mutant strain was silenced using the RNA interference technique. The mutant strain is motile, aggregated, and could not undergo further morphological development. The growth rate is high and the cells show a shortened cell cycle comparing with wild-type D. discoideum. However, the mechanisms regarding these actions remain unclear. mRNA differential display was used in this study to identify genetic differences. A novel D. discoideum gene (GenBank accession number: KC759140) encoding a new zinc protease was cloned. The amino acid sequence of the novel gene exhibited a conserved zinc-binding domain (HEX2HX18E) that allowed its classification into the M1 family of metallopeptidases. The gene encoded a 345-amino acid protein with a theoretical molecular mass of 39.69 kDa and a theoretical pI of 6.05. This protein showed strong homology with leukotriene A4 (LTA4) hydrolase of Homo sapiens (41% identity and 60% similarity at the amino acid level). By analyzing quantitative reverse transcription-polymerase chain reaction data, this zinc protease gene was more highly expressed in D. discoideum allC RNAi mutant type than in wild-type KAx-3 cells during the trophophase. The novel zinc protease gene may function as an LTA4 hydrolase and contribute to the shortening of the allC RNAi mutant cell cycle.

  17. The calcineurin dependent transcription factor TacA is involved in development and the stress response of Dictyostelium discoideum.

    PubMed

    Thewes, Sascha; Krohn, Stefanie; Schmith, Anika; Herzog, Sergej; Stach, Thomas; Weissenmayer, Barbara; Mutzel, Rupert

    2012-10-01

    Calcineurin is an important signalling protein in a plethora of Ca(2+)-regulated cellular processes. In contrast to what is known about the function of calcineurin in various organisms, information on calcineurin substrates is still limited. Here we describe the identification and characterisation of the transcription factor activated by calcineurin (TacA) in the model organism Dictyostelium discoideum. TacA is a putative zinc-finger transcription factor orthologue of yeast Crz1. In resting unstimulated cells the protein is located in the cytosol and translocates to the nucleus in a calcineurin-dependent manner after Ca(2+)-stimulation. Nuclear export of TacA is partially dependent on GskA, the Dictyostelium orthologue of mammalian GSK3. The expression of tacA is developmentally regulated with its kinetics roughly paralleling calcineurin regulation. Silencing of tacA via RNAi leads to developmental defects and dysregulation of developmentally regulated and Ca(2+)-regulated marker genes. Additionally, TacA is involved in the stress response of D. discoideum during development in a separate pathway to the well-known stress response in Dictyostelium via STATc. Finally we provide evidence that TacA is not only an orthologue of yeast Crz1 but also functionally related to mammalian NFAT.

  18. Reconstitution of functional eukaryotic ribosomes from Dictyostelium discoideum ribosomal proteins and RNA.

    PubMed

    Mangiarotti, G; Chiaberge, S

    1997-08-08

    40 and 60 S ribosomal subunits have been reconstituted in vitro from purified ribosomal RNA and ribosomal proteins of Dictyostelium discoideum. The functionality of the reconstituted ribosomes was demonstrated in in vitro mRNA-directed protein synthesis. The reassembly proceeded well with immature precursors of ribosomal RNA but poorly if at all with mature cytoplasmic RNA species. Reassembly also required a preparation of small nuclear RNA(s), acting as morphopoietic factor(s).

  19. Flow-driven waves and sink-driven oscillations during aggregation of Dictyostelium discoideum

    NASA Astrophysics Data System (ADS)

    Gholami, Azam; Zykov, Vladimir; Steinbock, Oliver; Bodenschatz, Eberhard

    The slime mold Dictyostelium discoideum (D.d) is a well-known model system for the study of biological pattern formation. Under starvation, D.d. cells aggregate chemotactically towards cAMP signals emitted periodically from an aggregation center. In the natural environment, D.d cells may experience fluid flows that can profoundly change the underlying wave generation process. We investigate spatial-temporal dynamics of a uniformly distributed population of D.d. cells in a flow-through narrow microfluidic channel with a cell-free inlet area. We show that flow can significantly influence the dynamics of the system and lead to a flow- driven instability that initiate downstream traveling cAMP waves. We also show that cell-free boundary regions have a significant effect on the observed patterns and can lead to a new kind of instability. Since there are no cells in the inlet to produce cAMP, the points in the vicinity of the inlet lose cAMP due to advection or diffusion and gain only a little from the upstream of the channel (inlet). In other words, there is a large negative flux of cAMP in the neighborhood close to the inlet, which can be considered as a sink. This negative flux close to the inlet drives a new kind of instability called sink-driven oscillations. Financial support of the MaxSynBio Consortium is acknowledged.

  20. Insertion of transformation vector DNA into different chromosomal sites of Dictyostelium discoideum as determined by pulse field electrophoresis.

    PubMed

    Cole, R A; Williams, K L

    1988-06-10

    Chromosomes of the cellular slime mold Dictyostelium discoideum were fractionated on three pulse field gel electrophoresis systems (pulse field, orthogonal field and C.H.E.F. (Contour-clamped Homogeneous Electric Fields] into a series of 13 bands ranging from 0.1 Mb to over 2 Mb in size. Since this organism has only seven chromosomes (estimated to be 1-10 Mb), and -90 copies of an 88-kilobase linear ribosomal DNA molecule (14% of genome), it was apparent that not all of these bands were whole chromosomes. However these bands were reproducibly obtained with the cell preparation used. They fell into three categories: i) four large poorly resolved DNA molecules (-2 Mb in size) which represent very large fragments or intact chromosomes, ii) eight faint bands ranging from 0.1 Mb to 2 Mb, iii) a prominent band in the apparent size range of about 0.15 Mb. Cloned Fragment V of an EcoR1 digest of the ribosomal DNA, hybridized to the 0.15 Mb band indicating it contained the linear ribosomal DNA. This chromosomal banding pattern was used to examine the stability and location of vector DNA in 16 transformed strains of D. discoideum. Each transformed strain was initially selected on the basis of G418 resistance with an integrating vector containing pBR322 sequences. Eleven transformants still carried pBR322 sequences after more than 60 generations of growth without selection on G418. All four strains transformed with constructs containing regions of the D. discoideum plasmid Ddp1 had lost their pBR322 insert, indicating that integration of Dictyostelium plasmid DNA into chromosomes leads to instability. Orthogonal field electrophoresis of the eleven strains still carrying pBR322 sequences revealed at least seven different integrating sites for the transforming DNA. We conclude that these vectors have many possible sites of integration in the D. discoideum genome.

  1. Translational control of discoidin lectin expression in drsA suppressor mutants of Dictyostelium discoideum.

    PubMed Central

    Alexander, S; Leone, S; Ostermeyer, E

    1991-01-01

    Genetic analysis in Dictyostelium discoideum has identified regulatory genes which control the developmental expression of the discoidin lectin multigene family. Among these, the drsA mutation is a dominant second-site suppressor of another mutation, disB, which has the discoidinless phenotype. We now demonstrate a novel mechanism by which the drsA allele exerts its suppressive effect on the disB mutation. Interestingly, drsA does not merely bypass the disB mutation and restore the wild-type pattern of lectin expression. Rather, drsA mutant cells have high levels of discoidin lectin synthesis during growth but do not express lectins during aggregation. In contrast, wild-type cells only express lectin protein during the aggregation period of development. Phenocopies of the drsA mutation show a pattern of discoidin expression similar to that seen in the bona fide mutant. These data suggest that there may be a mechanism of negative feedback, resulting from the high levels of discoidin lectin made during growth, which inhibits further discoidin lectin expression during development. Northern (RNA) analysis of developing drsA mutant cells shows that these cells contain high levels of discoidin mRNA, although no discoidin lectin protein is being translated from these messages. Therefore, expression of the discoidin gene family can be controlled at the level of translation as well as transcription. Images PMID:2038325

  2. Phosphorylation of proteins in Dictyostelium discoideum during development

    SciTech Connect

    Coffman, D.S.

    1982-01-01

    The phosphoproteins in D. discoideum were studied with respect to their formation, metabolic stability, cellular and subcellular distribution. Special emphasis was on the role of cAMP on the pattern of phosphorylation. Amoebae were metabolically labeled with /sup 32/P/sub i/; subsequently proteins of the total lysate, nuclei and membranes were resolved by SDS-polyacrylamide gel electrophoresis and subjected to autoradiography. Numerous changes in the profile of phosphoproteins were observed during development. Functions were assigned to four membranal phosphoproteins; only one protein, the heavy chain of myosin, was susceptible to phosphorylation in vitro when purified membranes and /sup 32/P-ATP were used. A comparison between the time of protein synthesis and phosphorylation, as examined in vivo using /sup 35/S-methionine and /sup 32/P/sub i/ labeling of amoebae and two-dimensional gel electrophoresis, indicated that phosphorylation is concurrent with synthesis. It appears then that there are two classes of membranal phosphoproteins in D. discoideum which differ with respect to the stability of the phosphate moiety. It is evident that the turnover of the phosphate moiety in myosin heavy chain plays a crucial role in the function of myosin; a role for the metabolically inert phosphate of other membranal proteins remains to be established. The G protein which couples occupancy of hormone receptor to stimulation of adenylate cyclase in higher multicellular eukaryotes was detected in D. discoideum. The G protein is present in approximately equal amounts in vegetative and in developing amoebae.

  3. A large scale screen reveals genes that mediate electrotaxis in Dictyostelium discoideum**

    PubMed Central

    Gao, Runchi; Zhao, Siwei; Jiang, Xupin; Sun, Yaohui; Zhao, Sanjun; Gao, Jing; Borleis, Jane; Willard, Stacey; Tang, Ming; Cai, Huaqing; Kamimura, Yoichiro; Huang, Yuesheng; Jiang, Jianxin; Huang, Zunxi; Mogilner, Alex; Pan, Tingrui; Devreotes, Peter N; Zhao, Min

    2015-01-01

    Directional cell migration in an electric field, a phenomenon called galvanotaxis or electrotaxis, occurs in many types of cells, and may play an important role in wound healing and development. Small extracellular electric fields can guide the migration of amoeboid cells, and here, we established a large-scale screening approach to search for mutants with electrotaxis phenotypes from a collection of 563 Dictyostelium discoideum strains with morphological defects. We identified 28 strains that were defective in electrotaxis and 10 strains with a slightly higher directional response. Using plasmid rescue followed by gene disruption, we identified some of the mutated genes, including some previously implicated in chemotaxis. Amongst these we studied PiaA, which encodes a critical component of TORC2, a kinase protein complex that transduces changes in motility by activating the kinase PKB (also known as Akt). Furthermore, we found that electrotaxis was decreased in mutants lacking gefA, rasC, rip3, lst8 or pkbR1, genes that encode other components of the TORC2-PKB pathway. Thus, we have developed a high-throughput screening technique that will be a useful tool to elucidate the molecular mechanisms of electrotaxis. PMID:26012633

  4. Proteomic profiling of the extracellular matrix (slime sheath) of Dictyostelium discoideum.

    PubMed

    Huber, Robert J; O'Day, Danton H

    2015-10-01

    Dictyostelium discoideum has historically served as a model system for cell and developmental biology, but recently it has gained increasing attention as a model for the study of human diseases. The extracellular matrix (ECM) of this eukaryotic microbe serves multiple essential functions during development. It not only provides structural integrity to the moving multicellular pseudoplasmodium, or slug, it also provides components that regulate cell motility and differentiation. An LC/MS/MS analysis of slug ECM revealed the presence of a large number of proteins in two wild-type strains, NC4 and WS380B. GO annotation identified a large number of proteins involved in some form of binding (e.g. protein, polysaccharide, cellulose, carbohydrate, ATP, cAMP, ion, lipid, vitamin), as well as proteins that modulate metabolic processes, cell movement, and multicellular development. In addition, this proteomic analysis identified numerous expected (e.g. EcmA, EcmD, discoidin I, discoidin II), as well as unexpected (e.g. ribosomal and nuclear proteins) components. These topics are discussed in terms of the structure and function of the ECM during the development of this model amoebozoan and their relevance to ongoing biomedical research.

  5. Rates of synthesis and degradation of ribosomal ribonucleic acid during differentiation of Dictyostelium discoideum.

    PubMed Central

    Mangiarotti, G; Altruda, F; Lodish, H F

    1981-01-01

    Synthesis of ribosomes and ribosomal ribonucleic acid (RNA) continued during differentiation of Dictyostelium discoideum concurrently with extensive turnover of ribosomes synthesized during both growth and developmental stages. We show here that the rate of synthesis of 26S and 17S ribosomal RNA during differentiation was less than 15% of that in growing cells, and by the time of sorocarp formation only about 25% of the cellular ribosomes had been synthesized during differentiation. Ribosomes synthesized during growth and differentiation were utilized in messenger RNA translation to the same extent; about 50% of each class were on polyribosomes. Ribosome degradation is apparently an all-or-nothing process, since virtually all 80S monosomes present in developing cells could be incorporated into polysomes when growth conditions were restored. By several criteria, ribosomes synthesized during growth and differentiation were functionally indistinguishable. Our data, together with previously published information on changes in the messenger RNA population during differentiation, indicate that synthesis of new ribosomes is not necessary for translation of developmentally regulated messenger RNA. We also establish that the overall rate of messenger RNA synthesis during differentiation is less than 15% of that in growing cells. PMID:6965093

  6. Partial Purification and Characterization of Glycogen Phosphorylase from Dictyostelium discoideum1

    PubMed Central

    Jones, Theodore H. D.; Wright, Barbara E.

    1970-01-01

    Glycogen phosphorylase was isolated from cells of Dictyostelium discoideum in the culmination stage of development and purified 35-fold. The enzyme had a pH optimum of 6.9 and contained sulfhydryl groups essential for activity. The Km values for phosphate and glycogen were 3 mm and 0.06% (w/v), respectively. No dependence on, or stimulation by, any nucleotide was observed and a wide variety of nucleotides and glycolytic intermediates did not inhibit the enzyme. Nucleotide sugars competitively inhibited the enzyme. Guanosine diphosphoglucose and adenosine diphosphoglucose were the most effective, and uridine diphosphoglucose was the least effective of the nucleotide sugars tested. The specific activity of glycogen phosphorylase increased from about 0.004 unit per mg of protein in aggregating cells to about 0.024 unit per mg in culminating cells, and then decreased during sorocarp formation. This increase in enzyme specific activity during the starvation and aging of the system can account for the increased rate of glycogen degradation during this period of development. Amylase specific activity, measured at pH 4.8 and 6.9, varied between 0.005 and 0.013 unit per mg of protein during all stages of development. PMID:5530813

  7. Rates of synthesis and degradation of ribosomal ribonucleic acid during differentiation of Dictyostelium discoideum.

    PubMed

    Mangiarotti, G; Altruda, F; Lodish, H F

    1981-01-01

    Synthesis of ribosomes and ribosomal ribonucleic acid (RNA) continued during differentiation of Dictyostelium discoideum concurrently with extensive turnover of ribosomes synthesized during both growth and developmental stages. We show here that the rate of synthesis of 26S and 17S ribosomal RNA during differentiation was less than 15% of that in growing cells, and by the time of sorocarp formation only about 25% of the cellular ribosomes had been synthesized during differentiation. Ribosomes synthesized during growth and differentiation were utilized in messenger RNA translation to the same extent; about 50% of each class were on polyribosomes. Ribosome degradation is apparently an all-or-nothing process, since virtually all 80S monosomes present in developing cells could be incorporated into polysomes when growth conditions were restored. By several criteria, ribosomes synthesized during growth and differentiation were functionally indistinguishable. Our data, together with previously published information on changes in the messenger RNA population during differentiation, indicate that synthesis of new ribosomes is not necessary for translation of developmentally regulated messenger RNA. We also establish that the overall rate of messenger RNA synthesis during differentiation is less than 15% of that in growing cells.

  8. A cyanobacterial light activated adenylyl cyclase partially restores development of a Dictyostelium discoideum, adenylyl cyclase a null mutant.

    PubMed

    Chen, Zhi-Hui; Raffelberg, Sarah; Losi, Aba; Schaap, Pauline; Gärtner, Wolfgang

    2014-12-10

    A light-regulated adenylyl cyclase, mPAC, was previously identified from the cyanobacterium Microcoleus chthonoplastes PCC7420. MPAC consists of a flavin-based blue light-sensing LOV domain and a catalytic domain. In this work, we expressed mPAC in an adenylate cyclase A null mutant (aca-) of the eukaryote Dictyostelium discoideum and tested to what extent light activation of mPAC could restore the cAMP-dependent developmental programme of this organism. Amoebas of Dictyostelium, a well-established model organism, generate and respond to cAMP pulses, which cause them to aggregate and construct fruiting bodies. mPAC was expressed under control of a constitutive actin-15 promoter in D. discoideum and displayed low basal adenylyl cyclase activity in darkness that was about five-fold stimulated by blue light. mPAC expression in aca- cells marginally restored aggregation and fruiting body formation in darkness. However, more and larger fruiting bodies were formed when mPAC expressing cells were incubated in light. Extending former applications of light-regulated AC, these results demonstrate that mPAC can be used to manipulate multicellular development in eukaryotes in a light dependent manner.

  9. Relevant Genes Linked to Virulence Are Required for Salmonella Typhimurium to Survive Intracellularly in the Social Amoeba Dictyostelium discoideum

    PubMed Central

    Riquelme, Sebastián; Varas, Macarena; Valenzuela, Camila; Velozo, Paula; Chahin, Nicolás; Aguilera, Paulina; Sabag, Andrea; Labra, Bayron; Álvarez, Sergio A.; Chávez, Francisco P.; Santiviago, Carlos A.

    2016-01-01

    The social amoeba Dictyostelium discoideum has proven to be a useful model for studying relevant aspects of the host-pathogen interaction. In this work, D. discoideum was used as a model to study the ability of Salmonella Typhimurium to survive in amoebae and to evaluate the contribution of selected genes in this process. To do this, we performed infection assays using axenic cultures of D. discoideum co-cultured with wild-type S. Typhimurium and/or defined mutant strains. Our results confirmed that wild-type S. Typhimurium is able to survive intracellularly in D. discoideum. In contrast, mutants ΔaroA and ΔwaaL are defective in intracellular survival in this amoeba. Next, we included in our study a group of mutants in genes directly linked to Salmonella virulence. Of note, mutants ΔinvA, ΔssaD, ΔclpV, and ΔphoPQ also showed an impaired ability to survive intracellularly in D. discoideum. This indicates that S. Typhimurium requires a functional biosynthetic pathway of aromatic compounds, a lipopolysaccharide containing a complete O-antigen, the type III secretion systems (T3SS) encoded in SPI-1 and SPI-2, the type VI secretion system (T6SS) encoded in SPI-6 and PhoP/PhoQ two-component system to survive in D. discoideum. To our knowledge, this is the first report on the requirement of O-antigen and T6SS in the survival of Salmonella within amoebae. In addition, mutants ΔinvA and ΔssaD were internalized in higher numbers than the wild-type strain during competitive infections, suggesting that S. Typhimurium requires the T3SS encoded in SPI-1 and SPI-2 to evade phagocytosis by D. discoideum. Altogether, these results indicate that S. Typhimurium exploits a common set of genes and molecular mechanisms to survive within amoeba and animal host cells. The use of D. discoideum as a model for host–pathogen interactions will allow us to discover the gene repertoire used by Salmonella to survive inside the amoeba and to study the cellular processes that are affected

  10. Relevant Genes Linked to Virulence Are Required for Salmonella Typhimurium to Survive Intracellularly in the Social Amoeba Dictyostelium discoideum.

    PubMed

    Riquelme, Sebastián; Varas, Macarena; Valenzuela, Camila; Velozo, Paula; Chahin, Nicolás; Aguilera, Paulina; Sabag, Andrea; Labra, Bayron; Álvarez, Sergio A; Chávez, Francisco P; Santiviago, Carlos A

    2016-01-01

    The social amoeba Dictyostelium discoideum has proven to be a useful model for studying relevant aspects of the host-pathogen interaction. In this work, D. discoideum was used as a model to study the ability of Salmonella Typhimurium to survive in amoebae and to evaluate the contribution of selected genes in this process. To do this, we performed infection assays using axenic cultures of D. discoideum co-cultured with wild-type S. Typhimurium and/or defined mutant strains. Our results confirmed that wild-type S. Typhimurium is able to survive intracellularly in D. discoideum. In contrast, mutants ΔaroA and ΔwaaL are defective in intracellular survival in this amoeba. Next, we included in our study a group of mutants in genes directly linked to Salmonella virulence. Of note, mutants ΔinvA, ΔssaD, ΔclpV, and ΔphoPQ also showed an impaired ability to survive intracellularly in D. discoideum. This indicates that S. Typhimurium requires a functional biosynthetic pathway of aromatic compounds, a lipopolysaccharide containing a complete O-antigen, the type III secretion systems (T3SS) encoded in SPI-1 and SPI-2, the type VI secretion system (T6SS) encoded in SPI-6 and PhoP/PhoQ two-component system to survive in D. discoideum. To our knowledge, this is the first report on the requirement of O-antigen and T6SS in the survival of Salmonella within amoebae. In addition, mutants ΔinvA and ΔssaD were internalized in higher numbers than the wild-type strain during competitive infections, suggesting that S. Typhimurium requires the T3SS encoded in SPI-1 and SPI-2 to evade phagocytosis by D. discoideum. Altogether, these results indicate that S. Typhimurium exploits a common set of genes and molecular mechanisms to survive within amoeba and animal host cells. The use of D. discoideum as a model for host-pathogen interactions will allow us to discover the gene repertoire used by Salmonella to survive inside the amoeba and to study the cellular processes that are affected

  11. Different mRNAs have different nuclear transit times in Dictyostelium discoideum aggregates.

    PubMed Central

    Mangiarotti, G; Zuker, C; Chisholm, R L; Lodish, H F

    1983-01-01

    Nuclear processing of mRNA precursors in differentiating multicellular Dictyostelium discoideum aggregates is markedly slower than in growing amoebae. Thus, we have been able to determine the time of nuclear processing of individual mRNA species in postaggregating cells by following the incorporation of 32PO4 into nuclear and cytoplasmic RNA complementary to cloned cDNAs. Precursors of mRNAs synthesized during both growth and differentiation remain in the nucleus for about 25 to 60 min. By contrast, typical mRNAs which are synthesized only by postaggregative cells have nuclear processing times between 50 and 100 min. Depending on the particular mRNA, between 20 and 60% of nuclear transcripts are converted into cytoplasmic mRNA. A third class of mRNAs are transcribed from a set of repetitive DNA segments and are expressed predominantly during differentiation. Nuclear precursors of these mRNAs are extensively degraded within the nucleus or very rapidly after transport to the cytoplasm. Those sequences that are stable in the cytoplasm exit from the nucleus only after a lag of over 2 h. Thus, mRNAs encoded by different genes that are subject to different types of developmental controls display different times of transit to the cytoplasm and different efficiencies of nuclear processing. Differential nuclear processing may contribute to the regulation of the level of individual cytoplasmic mRNAs. Images PMID:6621537

  12. Functional characterisation of parvulin-type peptidyl prolyl cis-trans isomerase, PinA in Dictyostelium discoideum.

    PubMed

    Haokip, Nemneineng; Naorem, Aruna

    2017-01-08

    Pin1-type parvulins are unique among PPIases that can catalyse an otherwise slow cis-trans isomerisation of phosphorylated peptide bond preceding proline in target proteins. This prolyl isomerisation process can regulate activity, stability and localisation of target proteins and thus control cellular processes like eukaryotic cell proliferation, cell cycle progression and gene regulation. Towards understanding the function of Pin1-type prolyl isomerisation in Dictyostelium discoideum, a slime mould with distinct growth and developmental phases, we identified PinA as a novel Pin1-type parvulin by its ability to complement the temperature sensitivity phenotype associated with a mutation in ESS1 in S. cerevisiae. In D. discoideum, pinA is temporally and spatially regulated during growth and development. PinA is both nuclear as well as cytoplasmic in the growing cells. We further show that loss of pinA (pinA(-)) leads to decreased growth rate, reduced spore formation and abnormal prespore-prestalk patterning. We conclude that PinA is required for normal growth as well as development in D. discoideum.

  13. Conserved protein domains in a myosin heavy chain gene from Dictyostelium discoideum.

    PubMed Central

    Warrick, H M; De Lozanne, A; Leinwand, L A; Spudich, J A

    1986-01-01

    The 2116-amino acid myosin heavy chain sequence from Dictyostelium discoideum was determined from DNA sequence analysis of the cloned gene. The gene product can be divided into two distinct regions, a globular head region and a long alpha-helical, rod-like tail. In comparisons with nematode and mammalian muscle myosins, specific areas of the head region are highly conserved. These areas presumably reflect conserved functional and structural domains. Certain features that are present in the head region of nematode and mammalian muscle myosins, and that have been assumed to be important for myosin function, are missing in the Dictyostelium myosin sequence. The protein sequence of the Dictyostelium tail region is very poorly conserved with respect to the other myosins but displays the periodicities similar to those of muscle myosins. These periodicities are believed to play a role in filament formation. The 196-residue repeating unit that determines the 14.3-nm repeat seen in muscle thick filaments, the 28-residue charge repeating unit, and the 1,4 hydrophobic repeat previously described for the nematode myosin are all present in the Dictyostelium myosin rod sequence, suggesting that the filament structures of muscle and Dictyostelium myosins must be similar. PMID:3540939

  14. Identification of a cyclase-associated protein (CAP) homologue in Dictyostelium discoideum and characterization of its interaction with actin.

    PubMed

    Gottwald, U; Brokamp, R; Karakesisoglou, I; Schleicher, M; Noegel, A A

    1996-02-01

    In search for novel actin binding proteins in Dictyostelium discoideum we have isolated a cDNA clone coding for a protein of approximately 50 kDa that is highly homologous to the class of adenylyl cyclase-associated proteins (CAP). In Saccharomyces cerevisiae the amino-terminal part of CAP is involved in the regulation of the adenylyl cyclase whereas the loss of the carboxyl-terminal domain results in morphological and nutritional defects. To study the interaction of Dictyostelium CAP with actin, the complete protein and its amino-terminal and carboxyl-terminal domains were expressed in Escherichia coli and used in actin binding assays. CAP sequestered actin in a Ca2+ independent way. This activity was localized to the carboxyl-terminal domain. CAP and its carboxyl-terminal domain led to a fluorescence enhancement of pyrene-labeled G-actin up to 50% indicating a direct interaction, whereas the amino-terminal domain did not enhance. In polymerization as well as in viscometric assays the ability of the carboxyl-terminal domain to sequester actin and to prevent F-actin formation was approximately two times higher than that of intact CAP. The sequestering activity of full length CAP could be inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2), whereas the activity of the carboxyl-terminal domain alone was not influenced, suggesting that the amino-terminal half of the protein is required for the PIP2 modulation of the CAP function. In profilin-minus cells the CAP concentration is increased by approximately 73%, indicating that CAP may compensate some profilin functions in vivo. In migrating D. discoideum cells CAP was enriched at anterior and posterior plasma membrane regions. Only a weak staining of the cytoplasm was observed. In chemotactically stimulated cells the protein was very prominent in leading fronts. The data suggest an involvement of D. discoideum CAP in microfilament reorganization near the plasma membrane in a PIP2-regulated manner.

  15. [3H]Methotrexate as a ligand for the folate receptor of Dictyostelium discoideum.

    PubMed Central

    Nandini-Kishore, S G; Frazier, W A

    1981-01-01

    Studies of the folate chemotactic receptor of vegetative Dictyostelium discoideum cells have been hampered by the presence of the degradative enzyme folate deaminase. The diaminopterin compounds aminopterin and methotrexate (MTX) are chemoattractants but are not attacked by the deaminase. [3',5',7,9-3H]methotrexate ([3H]MTX) is a nondegraded radioligand for the folate receptor. Binding to the receptor is rapid, reaching steady state in less than one min, and reversible in less than 15 s by an excess of unlabeled MTX. A single class of binding sites is found with a Kd of 2 x 10(-8) M, which correlates well with the concentration dependence of chemotaxis. Folate, aminopterin, and MTX all compete for [3H]MTX binding, whereas pterin, p-aminobenzoate, and nucleotides do not. Analysis of the receptor during differentiation indicates a decrease in site number by a factor of 3 with no change in affinity during the first 7 hr. During this time, the directional response (chemotaxis) to MTX and folate is lost, but a nondirectional stimulation of motility rate (chemokinesis) is retained. The response to cyclic AMP displays reciprocal behavior, first appearing as a chemokinetic response and then as a chemotactic response. PMID:6278468

  16. Cooperation Induces Other Cooperation: Fruiting Bodies Promote the Evolution of Macrocysts in Dictyostelium discoideum.

    PubMed

    Shibasaki, Shota; Shirokawa, Yuka; Shimada, Masakazu

    2017-04-03

    Biological studies of the evolution of cooperation are challenging because this process is vulnerable to cheating. Many mechanisms, including kin discrimination, spatial structure, or by-products of self-interested behaviors, can explain this evolution. Here we propose that the evolution of cooperation can be induced by other cooperation. To test this idea, we used a model organism Dictyostelium discoideum because it exhibits two cooperative dormant phases, the fruiting body and the macrocyst. In both phases, the same chemoattractant, cyclic AMP (cAMP), is used to collect cells. This common feature led us to hypothesize that the evolution of macrocyst formation would be induced by coexistence with fruiting bodies. Before forming a mathematical model, we confirmed that macrocysts coexisted with fruiting bodies, at least under laboratory conditions. Next, we analyzed our evolutionary game theory-based model to investigate whether coexistence with fruiting bodies would stabilize macrocyst formation. The model suggests that macrocyst formation represents an evolutionarily stable strategy and a global invader strategy under this coexistence, but is unstable if the model ignores the fruiting body formation. This result indicates that the evolution of macrocyst formation and maintenance is attributable to coexistence with fruiting bodies. Therefore, macrocyst evolution can be considered as an example of evolution of cooperation induced by other cooperation.

  17. Identifying the molecular basis of functions in the transcriptome of the social amoeba Dictyostelium discoideum.

    PubMed

    Whitney, T J; Gardner, D G; Mott, M L; Brandon, M

    2010-03-09

    The unusual life cycle of Dictyostelium discoideum, in which an extra-cellular stressor such as starvation induces the development of a multicellular fruiting body consisting of stalk cells and spores from a culture of identical amoebae, provides an excellent model for investigating the molecular control of differentiation and the transition from single- to multi-cellular life, a key transition in development. We utilized serial analysis of gene expression (SAGE), a molecular method that is unbiased by dependence on previously identified genes, to obtain a transcriptome from a high-density culture of amoebae, in order to examine the transition to multi-cellular development. The SAGE method provides relative expression levels, which allows us to rank order the expressed genes. We found that a large number of ribosomal proteins were expressed at high levels, while various components of the proteosome were expressed at low levels. The only identifiable transmembrane signaling system components expressed in amoebae are related to quorum sensing, and their expression levels were relatively low. The most highly expressed gene in the amoeba transcriptome, dutA untranslated RNA, is a molecule with unknown function that may serve as an inhibitor of translation. These results suggest that high-density amoebae have not initiated development, and they also suggest a mechanism by which the transition into the development program is controlled.

  18. A GPCR involved in post aggregation events in Dictyostelium discoideum.

    PubMed

    Prabhu, Yogikala; Mondal, Subhanjan; Eichinger, Ludwig; Noegel, Angelika A

    2007-12-01

    Dictyostelium has 55 genes encoding seven-transmembrane G-protein-coupled receptors (GPCR) that belong to five of the six GPCR families. GrlA is one of the 17 family 3 GPCRs in Dictyostelium all of which resemble GABA(B) receptors from higher eukaryotes. GrlA is a 90-kDa protein present on the plasma membrane and on membranes of the ER. It has a large extracellular domain with homology to bacterial periplasmic proteins. The GrlA message is present throughout development and shows increased levels during the post aggregation stages. Inactivation of the grlA gene does not severely affect the growth phase, however, it leads to a delay in the development at the post aggregation stage. GrlA deficient strains show an altered DIF-1 response specific to the prestalk-specific ecmA and ecmB gene, reduced car2 and pkaC transcript levels and form a reduced number of spores. Germination of the spores was as in wild type. Transcriptional profiling supported the defect in the sporulation pathway as a large number of genes involved in the biogenesis and organization of the extracellular matrix and the sporulation process were significantly downregulated in the mutant.

  19. Clathrin heavy chain functions in sorting and secretion of lysosomal enzymes in Dictyostelium discoideum

    PubMed Central

    1994-01-01

    The clathrin heavy chain is a major component of clathrin-coated vesicles that function in selective membrane traffic in eukaryotic cells. We disrupted the clathrin heavy chain gene (chcA) in Dictyostelium discoideum to generate a stable clathrin heavy chain- deficient cell line. Measurement of pinocytosis in the clathrin-minus mutant revealed a four-to five-fold deficiency in the internalization of fluid-phase markers. Once internalized, these markers recycled to the cell surface of mutant cells at wild-type rates. We also explored the involvement of clathrin heavy chain in the trafficking of lysosomal enzymes. Pulse chase analysis revealed that clathrin-minus cells processed most alpha-mannosidase to mature forms, however, approximately 20-25% of the precursor molecules remained uncleaved, were missorted, and were rapidly secreted by the constitutive secretory pathway. The remaining intracellular alpha-mannosidase was successfully targeted to mature lysosomes. Standard secretion assays showed that the rate of secretion of alpha-mannosidase was significantly less in clathrin-minus cells compared to control cells in growth medium. Interestingly, the secretion rates of another lysosomal enzyme, acid phosphatase, were similar in clathrin-minus and wild-type cells. Like wild-type cells, clathrin-minus mutants responded to starvation conditions with increased lysosomal enzyme secretion. Our study of the mutant cells provide in vivo evidence for roles for the clathrin heavy chain in (a) the internalization of fluid from the plasma membrane; (b) sorting of hydrolase precursors from the constitutive secretory pathway to the lysosomal pathway; and (c) secretion of mature hydrolases from lysosomes to the extracellular space. PMID:8034739

  20. Loss of Cln3 function in the social amoeba Dictyostelium discoideum causes pleiotropic effects that are rescued by human CLN3.

    PubMed

    Huber, Robert J; Myre, Michael A; Cotman, Susan L

    2014-01-01

    The neuronal ceroid lipofuscinoses (NCL) are a group of inherited, severe neurodegenerative disorders also known as Batten disease. Juvenile NCL (JNCL) is caused by recessive loss-of-function mutations in CLN3, which encodes a transmembrane protein that regulates endocytic pathway trafficking, though its primary function is not yet known. The social amoeba Dictyostelium discoideum is increasingly utilized for neurological disease research and is particularly suited for investigation of protein function in trafficking. Therefore, here we establish new overexpression and knockout Dictyostelium cell lines for JNCL research. Dictyostelium Cln3 fused to GFP localized to the contractile vacuole system and to compartments of the endocytic pathway. cln3- cells displayed increased rates of proliferation and an associated reduction in the extracellular levels and cleavage of the autocrine proliferation repressor, AprA. Mid- and late development of cln3- cells was precocious and cln3- slugs displayed increased migration. Expression of either Dictyostelium Cln3 or human CLN3 in cln3- cells suppressed the precocious development and aberrant slug migration, which were also suppressed by calcium chelation. Taken together, our results show that Cln3 is a pleiotropic protein that negatively regulates proliferation and development in Dictyostelium. This new model system, which allows for the study of Cln3 function in both single cells and a multicellular organism, together with the observation that expression of human CLN3 restores abnormalities in Dictyostelium cln3- cells, strongly supports the use of this new model for JNCL research.

  1. A Novel Glycolipid Biosurfactant Confers Grazing Resistance upon Pantoea ananatis BRT175 against the Social Amoeba Dictyostelium discoideum

    PubMed Central

    Smith, Derek D. N.; Nickzad, Arvin

    2016-01-01

    ABSTRACT Pantoea is a versatile genus of bacteria with both plant- and animal-pathogenic strains, some of which have been suggested to cause human infections. There is, however, limited knowledge on the potential determinants used for host association and pathogenesis in animal systems. In this study, we used the model host Dictyostelium discoideum to show that isolates of Pantoea ananatis exhibit differential grazing susceptibility, with some being resistant to grazing by the amoebae. We carried out a high-throughput genetic screen of one grazing-resistant isolate, P. ananatis BRT175, using the D. discoideum pathosystem to identify genes responsible for the resistance phenotype. Among the 26 candidate genes involved in grazing resistance, we identified rhlA and rhlB, which we show are involved in the biosynthesis of a biosurfactant that enables swarming motility in P. ananatis BRT175. Using liquid chromatography-mass spectrometry (LC-MS), the biosurfactant was shown to be a glycolipid with monohexose-C10-C10 as the primary congener. We show that this novel glycolipid biosurfactant is cytotoxic to the amoebae and is capable of compromising cellular integrity, leading to cell lysis. The production of this biosurfactant may be important for bacterial survival in the environment and could contribute to the establishment of opportunistic infections. IMPORTANCE The genetic factors used for host interaction by the opportunistic human pathogen Pantoea ananatis are largely unknown. We identified two genes that are important for the production of a biosurfactant that confers grazing resistance against the social amoeba Dictyostelium discoideum. We show that the biosurfactant, which exhibits cytotoxicity toward the amoebae, is a glycolipid that incorporates a hexose rather than rhamnose. The production of this biosurfactant may confer a competitive advantage in the environment and could potentially contribute to the establishment of opportunistic infections. PMID

  2. The phylogeny of the aromatic amino acid hydroxylases revisited by characterizing phenylalanine hydroxylase from Dictyostelium discoideum.

    PubMed

    Siltberg-Liberles, Jessica; Steen, Ida Helene; Svebak, Randi M; Martinez, Aurora

    2008-12-31

    The social amoeba Dictyostelium discoideum contains only one aromatic amino acid hydroxylase (AAAH) gene compared to at least three in metazoans. As shown in this work this gene codes for a phenylalanine hydroxylase (DictyoPAH) and phylogenetic analysis places this enzyme close to the precursor AAAHs, aiding to define the evolutionary history of the AAAH family. DictyoPAH shows significant similarities to other eukaryote PAH, but it exhibits higher activity with tetrahydrodictyopterin (DH4) than with tetrahydrobiopterin (BH4) as cofactor. DH4 is an abundant tetrahydropterin in D. discoideum while BH4 is the natural cofactor of the AAAHs in mammals. Moreover, DictyoPAH is devoid of the characteristic regulatory mechanisms of mammalian PAH such as positive cooperativity for L-Phe and activation by preincubation with the substrate. Analysis of the few active site substitutions between DictyoPAH and mammalian PAH, including mutant expression analysis, reveals potential structural determinants for allosteric regulation.

  3. Imaging G-protein coupled receptor (GPCR)-mediated signaling events that control chemotaxis of Dictyostelium discoideum.

    PubMed

    Xu, Xuehua; Jin, Tian

    2011-09-20

    Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly (1). This guided cell migration is referred as chemotaxis, which is essential for various cells to carry out their functions such as trafficking of immune cells and patterning of neuronal cells (2, 3). A large family of G-protein coupled receptors (GPCRs) detects variable small peptides, known as chemokines, to direct cell migration in vivo (4). The final goal of chemotaxis research is to understand how a GPCR machinery senses chemokine gradients and controls signaling events leading to chemotaxis. To this end, we use imaging techniques to monitor, in real time, spatiotemporal concentrations of chemoattractants, cell movement in a gradient of chemoattractant, GPCR mediated activation of heterotrimeric G-protein, and intracellular signaling events involved in chemotaxis of eukaryotic cells (5-8). The simple eukaryotic organism, Dictyostelium discoideum, displays chemotaxic behaviors that are similar to those of leukocytes, and D. discoideum is a key model system for studying eukaryotic chemotaxis. As free-living amoebae, D. discoideum cells divide in rich medium. Upon starvation, cells enter a developmental program in which they aggregate through cAMP-mediated chemotaxis to form multicullular structures. Many components involved in chemotaxis to cAMP have been identified in D. discoideum. The binding of cAMP to a GPCR (cAR1) induces dissociation of heterotrimeric G-proteins into Gγ and Gβγ subunits (7, 9, 10). Gβγ subunits activate Ras, which in turn activates PI3K, converting PIP(2;) into PIP(3;) on the cell membrane (11-13). PIP(3;) serve as binding sites for proteins with pleckstrin Homology (PH) domains, thus recruiting these proteins to the membrane (14, 15). Activation of cAR1 receptors also controls the membrane associations of PTEN, which dephosphorylates PIP(3;) to PIP(2;)(16, 17). The molecular mechanisms are evolutionarily conserved in

  4. The Dictyostelium discoideum cellulose synthase: Structure/function analysis and identification of interacting proteins

    SciTech Connect

    Richard L. Blanton

    2004-02-19

    OAK-B135 The major accomplishments of this project were: (1) the initial characterization of dcsA, the gene for the putative catalytic subunit of cellulose synthase in the cellular slime mold Dictyostelium discoideum; (2) the detection of a developmentally regulated event (unidentified, but perhaps a protein modification or association with a protein partner) that is required for cellulose synthase activity (i.e., the dcsA product is necessary, but not sufficient for cellulose synthesis); (3) the continued exploration of the developmental context of cellulose synthesis and DcsA; (4) the isolation of a GFP-DcsA-expressing strain (work in progress); and (5) the identification of Dictyostelium homologues for plant genes whose products play roles in cellulose biosynthesis. Although our progress was slow and many of our results negative, we did develop a number of promising avenues of investigation that can serve as the foundation for future projects.

  5. Characterization of a 1,4-. beta. -D-glucan synthase from Dictyostelium discoideum

    SciTech Connect

    Blanton, R.L.

    1992-01-15

    Various aspects of research concerning Dictyostelium discoideum are presented. The initial focus of this project was upon: the characterization of potential probes for the cellulose synthase (antibody and nucleic acid), the determination of the cultural induction conditions of cellulose synthesis, the solubilization of the enzyme activity, the development of a non-inhibitory disruption buffer, the generation and isolation of mutant strains deficient in cellulose synthesis, and the development of the capability to determine the degree of polymerization of the in vitro product. I have briefly summarized our most significant findings with only selected data sets being shown in this report in the interest of brevity.

  6. Regulation of Spatiotemporal Patterns by Biological Variability: General Principles and Applications to Dictyostelium discoideum

    PubMed Central

    Grace, Miriam; Hütt, Marc-Thorsten

    2015-01-01

    Spatiotemporal patterns often emerge from local interactions in a self-organizing fashion. In biology, the resulting patterns are also subject to the influence of the systematic differences between the system’s constituents (biological variability). This regulation of spatiotemporal patterns by biological variability is the topic of our review. We discuss several examples of correlations between cell properties and the self-organized spatiotemporal patterns, together with their relevance for biology. Our guiding, illustrative example will be spiral waves of cAMP in a colony of Dictyostelium discoideum cells. Analogous processes take place in diverse situations (such as cardiac tissue, where spiral waves occur in potentially fatal ventricular fibrillation) so a deeper understanding of this additional layer of self-organized pattern formation would be beneficial to a wide range of applications. One of the most striking differences between pattern-forming systems in physics or chemistry and those in biology is the potential importance of variability. In the former, system components are essentially identical with random fluctuations determining the details of the self-organization process and the resulting patterns. In biology, due to variability, the properties of potentially very few cells can have a driving influence on the resulting asymptotic collective state of the colony. Variability is one means of implementing a few-element control on the collective mode. Regulatory architectures, parameters of signaling cascades, and properties of structure formation processes can be "reverse-engineered" from observed spatiotemporal patterns, as different types of regulation and forms of interactions between the constituents can lead to markedly different correlations. The power of this biology-inspired view of pattern formation lies in building a bridge between two scales: the patterns as a collective state of a very large number of cells on the one hand, and the internal

  7. Chemoattraction and chemotaxis in Dictyostelium discoideum: myxamoeba cannot read spatial gradients of cyclic adenosine monophosphate.

    PubMed

    Vicker, M G; Schill, W; Drescher, K

    1984-06-01

    Myxamoebae of the morphogenetic cellular slime mold Dictyostelium discoideum are thought to be able to accurately read and respond to directional information in spatial gradients of cyclic AMP. We examined the spatial and temporal mechanisms proposed for chemotaxis by comparing the behavior of spreading or evenly distributed cell populations after exposure to well-defined spatial gradients. The effects of gradient generation on cells were avoided by using predeveloped gradients. Qualitatively different responses were obtained using (a) isotropic, (b) static spatial, or (c) temporal (impulse) gradients in a simple chamber of penetrable micropore filters. We simulated models of chemotaxis and chemokinesis to aid our interpretations. The attractive and locomotory responses of populations were maximally stimulated by 0.05 microM cyclic AMP, provided that cellular phosphodiesterase was inhibited. But a single impulse of cyclic AMP during gradient development caused a greater and qualitatively different attraction. Attraction in spatial gradients was only transient, in that populations eventually developed a random distribution when confined to a narrow territory. Populations never accumulated nor lost their random distribution even in extremely steep spatial gradients. Attraction in spatial gradients was inducible only in spreading populations, not randomly distributed ones. Thus, spatial gradients effect biased-random locomotion: i.e., chemokinesis without adaptation. Cells cannot read gradients; the reaction of the cells is stochastic. Spatial gradients do not cause chemotaxis, which probably requires a sharp stimulant concentration increase (a temporal gradient) as a pulse or impulse. The results also bear on concepts of how embryonic cells might be able to decipher the positional information in a morphogen spatial gradient during development.

  8. Chemoattraction and chemotaxis in Dictyostelium discoideum: myxamoeba cannot read spatial gradients of cyclic adenosine monophosphate

    PubMed Central

    1984-01-01

    Myxamoebae of the morphogenetic cellular slime mold Dictyostelium discoideum are thought to be able to accurately read and respond to directional information in spatial gradients of cyclic AMP. We examined the spatial and temporal mechanisms proposed for chemotaxis by comparing the behavior of spreading or evenly distributed cell populations after exposure to well-defined spatial gradients. The effects of gradient generation on cells were avoided by using predeveloped gradients. Qualitatively different responses were obtained using (a) isotropic, (b) static spatial, or (c) temporal (impulse) gradients in a simple chamber of penetrable micropore filters. We simulated models of chemotaxis and chemokinesis to aid our interpretations. The attractive and locomotory responses of populations were maximally stimulated by 0.05 microM cyclic AMP, provided that cellular phosphodiesterase was inhibited. But a single impulse of cyclic AMP during gradient development caused a greater and qualitatively different attraction. Attraction in spatial gradients was only transient, in that populations eventually developed a random distribution when confined to a narrow territory. Populations never accumulated nor lost their random distribution even in extremely steep spatial gradients. Attraction in spatial gradients was inducible only in spreading populations, not randomly distributed ones. Thus, spatial gradients effect biased-random locomotion: i.e., chemokinesis without adaptation. Cells cannot read gradients; the reaction of the cells is stochastic. Spatial gradients do not cause chemotaxis, which probably requires a sharp stimulant concentration increase (a temporal gradient) as a pulse or impulse. The results also bear on concepts of how embryonic cells might be able to decipher the positional information in a morphogen spatial gradient during development. PMID:6327727

  9. Translational control of ribosomal protein synthesis during early Dictyostelium discoideum development.

    PubMed Central

    Steel, L F; Jacobson, A

    1987-01-01

    Throughout the developmental program of Dictyostelium discoideum there are substantial changes in the rates of both ribosome utilization and rRNA transcription and processing. We examined the regulation of ribosomal protein (r-protein) gene expression and found that, at the start of development, expression of these genes was drastically and specifically reduced by a block to translational initiation. An apparently separate event signals a sudden decrease in the relative amount of r-protein mRNA at about 10 h of development, a time when aggregated amoebae are forming tight cell-cell contacts. For the first 9 h of development, the relative amount of r-protein mRNA remained essentially unchanged and comparable to levels detected in growing cells. While the r-protein mRNAs were almost fully loaded on polysomes during vegetative growth, they were specifically excluded from polysomes at the start of development. The translational block was not the result of irreversible structural changes which inactivate the r-protein mRNAs since they remained translatable both in vitro, in wheat germ extracts, and in vivo, where they were recruited onto polysomes in the presence of the elongation inhibitor cycloheximide. In addition, precise measurements of poly(A) tail lengths on individual hybrid-selected mRNA species showed that there is no difference in the poly(A) tail length of r-protein mRNA isolated from growing cells and 1-h developing cells. Therefore, changes in translational efficiency cannot be attributed to cleavage of poly(A) tails. Images PMID:2882416

  10. The Actinome of Dictyostelium discoideum in Comparison to Actins and Actin-Related Proteins from Other Organisms

    PubMed Central

    Joseph, Jayabalan M.; Fey, Petra; Ramalingam, Nagendran; Liu, Xiao I.; Rohlfs, Meino; Noegel, Angelika A.; Müller-Taubenberger, Annette; Glöckner, Gernot; Schleicher, Michael

    2008-01-01

    Actin belongs to the most abundant proteins in eukaryotic cells which harbor usually many conventional actin isoforms as well as actin-related proteins (Arps). To get an overview over the sometimes confusing multitude of actins and Arps, we analyzed the Dictyostelium discoideum actinome in detail and compared it with the genomes from other model organisms. The D. discoideum actinome comprises 41 actins and actin-related proteins. The genome contains 17 actin genes which most likely arose from consecutive gene duplications, are all active, in some cases developmentally regulated and coding for identical proteins (Act8-group). According to published data, the actin fraction in a D. discoideum cell consists of more than 95% of these Act8-type proteins. The other 16 actin isoforms contain a conventional actin motif profile as well but differ in their protein sequences. Seven actin genes are potential pseudogenes. A homology search of the human genome using the most typical D. discoideum actin (Act8) as query sequence finds the major actin isoforms such as cytoplasmic beta-actin as best hit. This suggests that the Act8-group represents a nearly perfect actin throughout evolution. Interestingly, limited data from D. fasciculatum, a more ancient member among the social amoebae, show different relationships between conventional actins. The Act8-type isoform is most conserved throughout evolution. Modeling of the putative structures suggests that the majority of the actin-related proteins is functionally unrelated to canonical actin. The data suggest that the other actin variants are not necessary for the cytoskeleton itself but rather regulators of its dynamical features or subunits in larger protein complexes. PMID:18612387

  11. Ammonium phosphate in sori of Dictyostelium discoideum promotes spore dormancy through stimulation of the osmosensor ACG.

    PubMed

    Cotter, D A; Dunbar, A J; Buconjic, S D; Wheldrake, J F

    1999-08-01

    The sori of Dictyostelium discoideum (strains SG1, SG2, NC4 and V12) contained more than 100 mM ammonium phosphate. Glutamine synthetase (GS), which could remove ammonia from the sorus, was not present in 2-d-old dormant spores but enzyme activity returned to vegetative levels after spore germination. Based on mRNA blotting, the activity of this enzyme in germinating spores appeared to be transcriptionally controlled. At the same time that GS activity was increasing, ammonia was released from germinating spores. Exogenous ammonium ions at a concentration of 28 mM did not block germination nor modulate GS activity in nascent amoebae. It was concluded that the transcription and translation of GS is not environmentally regulated but is an integral part of the germination process, preparing nascent amoebae for vegetative growth. An exogenous concentration of 69 mM ammonium phosphate could maintain dormancy in spores of strains SG1 and SG2 for at least a week in the absence of any other inhibitory component from the sori. The inhibition was reversible at any time either by dilution or by washing the spores free of the ammonium ion. Spores of strain acg- were not inhibited by 100 mM ammonium phosphate. A model is presented in which GS in prespore cells serves as a sink for ammonia to allow the osmotically sensitive adenylyl cyclase aggregation protein (ACA) to activate protein kinase A (PKA) to induce fruiting-body formation. After fruiting-body formation is complete, the decline in GS and ACA activities in developing spores is offset by their replacement with the osmotically and ammonia-stimulated adenylyl cyclase osmosensor for germination (ACG). Ammonia and discadenine may act as separate signals to synergistically activate PKA by stimulating ACG activity while inhibiting cAMP phosphodiestrase activity in fully dormant spores.

  12. Peptide: N- glycanase is expressed in prestalk cells and plays a role in the differentiation of prespore cells during development of Dictyostelium discoideum.

    PubMed

    Gosain, Anuradha; Srivastava, Anju; Saran, Shweta

    2014-03-01

    Peptide: N-glycanase (PNGase) enzyme is found throughout eukaryotes and plays an important role in the misfolded glycoprotein degradation pathway. This communication reports the expression patterns of the pngase transcript (as studied by the analysis of beta-galactosidase reporter driven by the putative pngase promoter) and protein (as studied by the analysis of beta-galactosidase reporter expressed under the putative pngase promoter as a fusion with the pngase ORF) during development and further elucidated the developmental defects of the cells lacking PNGase (png(-)). The results show that the DdPNGase is an essential protein expressed throughout development and beta-galactosidase activity was present in the anterior part of the slug. In structures derived from a null mutant for pngase, the prestalk A and AO patterning was expanded and covered a large section of the prespore region of the slugs. When developed as chimeras with wild type, the png(-) cells preferentially populate the prestalk/stalk region. When the mutants were mixed in higher ratios, they also tend to form the prespore/spore cells. The results emphasize that the DdPNGase has an essential role during development and the mutants have defects in a system that changes the physiological dynamics in the prespore cells. DdPNGase play a role in development both during aggregation and in the differentiation of prespore cells.

  13. Lipid Composition of Multilamellar Bodies Secreted by Dictyostelium discoideum Reveals Their Amoebal Origin

    PubMed Central

    Paquet, Valérie E.; Lessire, René; Domergue, Frédéric; Fouillen, Laetitia; Filion, Geneviève; Sedighi, Ahmadreza

    2013-01-01

    When they are fed with bacteria, Dictyostelium discoideum amoebae produce and secrete multilamellar bodies (MLBs), which are composed of membranous material. It has been proposed that MLBs are a waste disposal system that allows D. discoideum to eliminate undigested bacterial remains. However, the real function of MLBs remains unknown. Determination of the biochemical composition of MLBs, especially lipids, represents a way to gain information about the role of these structures. To allow these analyses, a protocol involving various centrifugation procedures has been developed to purify secreted MLBs from amoeba-bacterium cocultures. The purity of the MLB preparation was confirmed by transmission electron microscopy and by immunofluorescence using H36, an antibody that binds to MLBs. The lipid and fatty acid compositions of pure MLBs were then analyzed by high-performance thin-layer chromatography (HPTLC) and gas chromatography (GC), respectively, and compared to those of amoebae as well as bacteria used as a food source. While the bacteria were devoid of phosphatidylcholine (PC) and phosphatidylinositol (PI), these two polar lipid species were major classes of lipids in MLBs and amoebae. Similarly, the fatty acid composition of MLBs and amoebae was characterized by the presence of polyunsaturated fatty acids, while cyclic fatty acids were found only in bacteria. These results strongly suggest that the lipids constituting the MLBs originate from the amoebal metabolism rather than from undigested bacterial membranes. This opens the possibility that MLBs, instead of being a waste disposal system, have unsuspected roles in D. discoideum physiology. PMID:23748431

  14. Dictyostelium discoideum has a single diacylglycerol kinase gene with similarity to mammalian theta isoforms.

    PubMed Central

    De La Roche, Marc A; Smith, Janet L; Rico, Maribel; Carrasco, Silvia; Merida, Isabel; Licate, Lucila; Côté, Graham P; Egelhoff, Thomas T

    2002-01-01

    Diacylglycerol kinases (DGKs) phosphorylate the neutral lipid diacylglycerol (DG) to produce phosphatidic acid (PA). In mammalian systems DGKs are a complex family of at least nine isoforms that are thought to participate in down-regulation of DG-based signalling pathways and perhaps activation of PA-stimulated signalling events. We report here that the simple protozoan amoeba Dictyostelium discoideum appears to contain a single gene encoding a DGK enzyme. This gene, dgkA, encodes a deduced protein that contains three C1-type cysteine-rich repeats, a DGK catalytic domain most closely related to the theta subtype of mammalian DGKs and a C-terminal segment containing a proline/glutamine-rich region and a large aspargine-repeat region. This gene corresponds to a previously reported myosin II heavy chain kinase designated myosin heavy chain-protein kinase C (MHC-PKC), but our analysis clearly demonstrates that this protein does not, as suggested by earlier data, contain a protein kinase catalytic domain. A FLAG-tagged version of DgkA expressed in Dictyostelium displayed robust DGK activity. Earlier studies indicating that disruption of this locus alters myosin II assembly levels in Dictyostelium raise the intriguing possibility that DG and/or PA metabolism may play a role in controlling myosin II assembly in this system. PMID:12296770

  15. Mitochondrial tRNA 5′-Editing in Dictyostelium discoideum and Polysphondylium pallidum*

    PubMed Central

    Abad, Maria G.; Long, Yicheng; Kinchen, R. Dimitri; Schindel, Elinor T.; Gray, Michael W.; Jackman, Jane E.

    2014-01-01

    Mitochondrial tRNA (mt-tRNA) 5′-editing was first described more than 20 years ago; however, the first candidates for 5′-editing enzymes were only recently identified in a eukaryotic microbe (protist), the slime mold Dictyostelium discoideum. In this organism, eight of 18 mt-tRNAs are predicted to be edited based on the presence of genomically encoded mismatched nucleotides in their aminoacyl-acceptor stem sequences. Here, we demonstrate that mt-tRNA 5′-editing occurs at all predicted sites in D. discoideum as evidenced by changes in the sequences of isolated mt-tRNAs compared with the expected sequences encoded by the mitochondrial genome. We also identify two previously unpredicted editing events in which G-U base pairs are edited in the absence of any other genomically encoded mismatches. A comparison of 5′-editing in D. discoideum with 5′-editing in another slime mold, Polysphondylium pallidum, suggests organism-specific idiosyncrasies in the treatment of U-G/G-U pairs. In vitro activities of putative D. discoideum editing enzymes are consistent with the observed editing reactions and suggest an overall lack of tRNA substrate specificity exhibited by the repair component of the editing enzyme. Although the presence of terminal mismatches in mt-tRNA sequences is highly predictive of the occurrence of mt-tRNA 5′-editing, the variability in treatment of U-G/G-U base pairs observed here indicates that direct experimental evidence of 5′-editing must be obtained to understand the complete spectrum of mt-tRNA editing events in any species. PMID:24737330

  16. Identification of a cyclase-associated protein (CAP) homologue in Dictyostelium discoideum and characterization of its interaction with actin.

    PubMed Central

    Gottwald, U; Brokamp, R; Karakesisoglou, I; Schleicher, M; Noegel, A A

    1996-01-01

    In search for novel actin binding proteins in Dictyostelium discoideum we have isolated a cDNA clone coding for a protein of approximately 50 kDa that is highly homologous to the class of adenylyl cyclase-associated proteins (CAP). In Saccharomyces cerevisiae the amino-terminal part of CAP is involved in the regulation of the adenylyl cyclase whereas the loss of the carboxyl-terminal domain results in morphological and nutritional defects. To study the interaction of Dictyostelium CAP with actin, the complete protein and its amino-terminal and carboxyl-terminal domains were expressed in Escherichia coli and used in actin binding assays. CAP sequestered actin in a Ca2+ independent way. This activity was localized to the carboxyl-terminal domain. CAP and its carboxyl-terminal domain led to a fluorescence enhancement of pyrene-labeled G-actin up to 50% indicating a direct interaction, whereas the amino-terminal domain did not enhance. In polymerization as well as in viscometric assays the ability of the carboxyl-terminal domain to sequester actin and to prevent F-actin formation was approximately two times higher than that of intact CAP. The sequestering activity of full length CAP could be inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2), whereas the activity of the carboxyl-terminal domain alone was not influenced, suggesting that the amino-terminal half of the protein is required for the PIP2 modulation of the CAP function. In profilin-minus cells the CAP concentration is increased by approximately 73%, indicating that CAP may compensate some profilin functions in vivo. In migrating D. discoideum cells CAP was enriched at anterior and posterior plasma membrane regions. Only a weak staining of the cytoplasm was observed. In chemotactically stimulated cells the protein was very prominent in leading fronts. The data suggest an involvement of D. discoideum CAP in microfilament reorganization near the plasma membrane in a PIP2-regulated manner. Images PMID

  17. Cloning and characterization of the Dictyostelium discoideum rasG genomic sequences.

    PubMed

    Robbins, S M; Williams, J G; Spiegelman, G B; Weeks, G

    1992-02-28

    A Dictyostelium discoideum genomic DNA clone containing the ras-related gene, rasG was isolated using the rasG cDNA as a probe. The genomic clone encompasses the entire coding region of the gene and 1.5 kb of 5' flanking region. The rasG gene contains a single intron as determined by sequence comparison with the cDNA, whereas the highly related rasD gene contains three introns. Primer extension analysis showed that transcription of the rasG gene initiates at multiple sites. Sequence analysis of the 5' flanking region of the gene revealed a stretch of thymine residues upstream from the transcription start sites but there is no evidence for a TATA box sequence.

  18. rRNA maturation as a "quality" control step in ribosomal subunit assembly in Dictyostelium discoideum.

    PubMed

    Mangiarotti, G; Chiaberge, S; Bulfone, S

    1997-10-31

    In Dictyostelium discoideum, newly assembled ribosomal subunits enter polyribosomes while they still contain immature rRNA. rRNA maturation requires the engagement of the subunits in protein synthesis and leads to stabilization of their structure. Maturation of pre-17 S rRNA occurs only after the newly formed 40 S ribosomal particle has entered an 80 S ribosome and participated at least in the formation of one peptide bond or in one translocation event; maturation of pre-26 S rRNA requires the presence on the 80 S particle of a peptidyl-tRNA containing at least 6 amino acids. Newly assembled particles that cannot fulfill these requirements for structural reasons are disassembled into free immature rRNA and ribosomal proteins.

  19. Iron metabolism and resistance to infection by invasive bacteria in the social amoeba Dictyostelium discoideum.

    PubMed

    Bozzaro, Salvatore; Buracco, Simona; Peracino, Barbara

    2013-01-01

    Dictyostelium cells are forest soil amoebae, which feed on bacteria and proliferate as solitary cells until bacteria are consumed. Starvation triggers a change in life style, forcing cells to gather into aggregates to form multicellular organisms capable of cell differentiation and morphogenesis. As a soil amoeba and a phagocyte that grazes on bacteria as the obligate source of food, Dictyostelium could be a natural host of pathogenic bacteria. Indeed, many pathogens that occasionally infect humans are hosted for most of their time in protozoa or free-living amoebae, where evolution of their virulence traits occurs. Due to these features and its amenability to genetic manipulation, Dictyostelium has become a valuable model organism for studying strategies of both the host to resist infection and the pathogen to escape the defense mechanisms. Similarly to higher eukaryotes, iron homeostasis is crucial for Dictyostelium resistance to invasive bacteria. Iron is essential for Dictyostelium, as both iron deficiency or overload inhibit cell growth. The Dictyostelium genome shares with mammals many genes regulating iron homeostasis. Iron transporters of the Nramp (Slc11A) family are represented with two genes, encoding Nramp1 and Nramp2. Like the mammalian ortholog, Nramp1 is recruited to phagosomes and macropinosomes, whereas Nramp2 is a membrane protein of the contractile vacuole network, which regulates osmolarity. Nramp1 and Nramp2 localization in distinct compartments suggests that both proteins synergistically regulate iron homeostasis. Rather than by absorption via membrane transporters, iron is likely gained by degradation of ingested bacteria and efflux via Nramp1 from phagosomes to the cytosol. Nramp gene disruption increases Dictyostelium sensitivity to infection, enhancing intracellular growth of Legionella or Mycobacteria. Generation of mutants in other "iron genes" will help identify genes essential for iron homeostasis and resistance to pathogens.

  20. Dual role of cAMP and involvement of both G-proteins and ras in regulation of ERK2 in Dictyostelium discoideum.

    PubMed

    Knetsch, M L; Epskamp, S J; Schenk, P W; Wang, Y; Segall, J E; Snaar-Jagalska, B E

    1996-07-01

    Dictyostelium discoideum expresses two Extracellular signal Regulated Kinases, ERK1 and ERK2, which are involved in growth, multicellular development and regulation of adenylyl cyclase. Binding of extracellular cAMP to cAMP receptor 1, a G-protein coupled cell surface receptor, transiently stimulates phosphorylation, activation and nuclear translocation of ERK2. Activation of ERK2 by cAMP is dependent on heterotrimeric G-proteins, since activation of ERK2 is absent in cells lacking the Galpha4 subunit. The small G-protein rasD also activates ERK2. In cells overexpressing a mutated, constitutively active rasD, ERK2 activity is elevated prior to cAMP stimulation. Intracellular cAMP and cAMP-dependent protein kinase (PKA) are essential for adaptation of the ERK2 response. This report shows that multiple signalling pathways are involved in regulation of ERK2 activity in D.discoideum.

  1. Effects of Nickel, Chlorpyrifos and Their Mixture on the Dictyostelium discoideum Proteome

    PubMed Central

    Boatti, Lara; Robotti, Elisa; Marengo, Emilio; Viarengo, Aldo; Marsano, Francesco

    2012-01-01

    Mixtures of chemicals can have additive, synergistic or antagonistic interactions. We investigated the effects of the exposure to nickel, the organophosphate insecticide chlorpyrifos at effect concentrations (EC) of 25% and 50% and their binary mixture (Ec25 + EC25) on Dictyostelium discoideum amoebae based on lysosomal membrane stability (LMS). We treated D. discoideum with these compounds under controlled laboratory conditions and evaluated the changes in protein levels using a two-dimensional gel electrophoresis (2DE) proteomic approach. Nickel treatment at EC25 induced changes in 14 protein spots, 12 of which were down-regulated. Treatment with nickel at EC50 resulted in changes in 15 spots, 10 of which were down-regulated. Treatment with chlorpyrifos at EC25 induced changes in six spots, all of which were down-regulated; treatment with chlorpyrifos at EC50 induced changes in 13 spots, five of which were down-regulated. The mixture corresponding to EC25 of each compound induced changes in 19 spots, 13 of which were down-regulated. The data together reveal that a different protein expression signature exists for each treatment, and that only a few proteins are modulated in multiple different treatments. For a simple binary mixture, the proteomic response does not allow for the identification of each toxicant. The protein spots that showed significant differences were identified by mass spectrometry, which revealed modulations of proteins involved in metal detoxification, stress adaptation, the oxidative stress response and other cellular processes. PMID:23443088

  2. Separate nuclear genes encode cytosolic and mitochondrial nucleoside diphosphate kinase in Dictyostelium discoideum.

    PubMed

    Troll, H; Winckler, T; Lascu, I; Müller, N; Saurin, W; Véron, M; Mutzel, R

    1993-12-05

    We have previously isolated cDNA clones for the gip17 gene encoding the cytosolic nucleoside diphosphate (NDP) kinase from Dictyostelium discoideum, and partial cDNAs for guk, a second member of the NDP kinase gene family (Wallet, V., Mutzel, R., Troll, H., Barzu, O., Wurster, B., Véron, M., and Lacombe, M. L. (1990) J. Natl. Cancer Inst. 80, 1199-1202). We now characterize genomic DNA clones for both NDP kinase genes, and we show that guk defines a nuclear-encoded mitochondrial NDP kinase. Isolated D. discoideum mitochondria contain 3% of the total cellular NDP kinase activity. Antibodies which specifically recognize and inhibit the activity of either cytosolic or mitochondrial NDP kinase unambiguously distinguish between these activities. The nascent mitochondrial NDP kinase contains a presequence of 57 amino acids that is removed during import into the organelle as shown by determination of the NH2 terminus of the mature protein from mitochondria. The genes for mitochondrial and cytosolic NDP kinases contain four and two introns, respectively. The positions of the of the introns in the gene for the cytosolic enzyme match exactly the positions of the second and fourth introns in the coding region of its mitochondrial homologue. From these results we conclude that the isozymes diverged from a common ancestor, and we discuss possible phylogenetic pathways for the evolution of cytosolic and organelle NDP kinases.

  3. Isolation of an actin-binding protein from membranes of Dictyostelium discoideum

    PubMed Central

    1985-01-01

    We prepared a probe of radiolabeled, glutaraldehyde cross-linked filamentous actin (F-actin) to study binding of actin to membranes of Dictyostelium discoideum. The probe bound to membranes or detergent extracts of membranes with a high affinity and in a saturable manner. The binding could be reduced by boiling of either the actin probe or the membranes, or by addition of excess native F-actin, but not by addition of an equivalent amount of bovine serum albumin, to the assay. The probe labeled several proteins when used to overlay sodium dodecyl sulfate gels of Dictyostelium membranes. One of these labeled proteins was a 24,000-mol-wt protein (p24), which was soluble only in the presence of a high concentration of sodium deoxycholate (5%, wt/vol) at room temperature or above. The p24 was purified by selective detergent extraction and column chromatography. When tested in a novel two-phase binding assay, p24 bound both native monomeric actin (G-actin) and F- actin in a specific manner. In this assay, G-actin bound p24 with a submicromolar affinity. PMID:3972891

  4. Dictyostelium discoideum CenB Is a Bona Fide Centrin Essential for Nuclear Architecture and Centrosome Stability ▿

    PubMed Central

    Mana-Capelli, Sebastian; Gräf, Ralph; Larochelle, Denis A.

    2009-01-01

    Centrins are a family of proteins within the calcium-binding EF-hand superfamily. In addition to their archetypical role at the microtubule organizing center (MTOC), centrins have acquired multiple functionalities throughout the course of evolution. For example, centrins have been linked to different nuclear activities, including mRNA export and DNA repair. Dictyostelium discoideum centrin B is a divergent member of the centrin family. At the amino acid level, DdCenB shows 51% identity with its closest relative and only paralog, DdCenA. Phylogenetic analysis revealed that DdCenB and DdCenA form a well-supported monophyletic and divergent group within the centrin family of proteins. Interestingly, fluorescently tagged versions of DdCenB were not found at the centrosome (in whole cells or in isolated centrosomes). Instead, DdCenB localized to the nuclei of interphase cells. This localization disappeared as the cells entered mitosis, although Dictyostelium cells undergo a closed mitosis in which the nuclear envelope (NE) does not break down. DdCenB knockout cells exhibited aberrant nuclear architecture, characterized by enlarged and deformed nuclei and loss of proper centrosome-nucleus anchoring (observed as NE protrusions). At the centrosome, loss of DdCenB resulted in defects in the organization and morphology of the MTOC and supernumerary centrosomes and centrosome-related bodies. The multiple defects that the loss of DdCenB generated at the centrosome can be explained by its atypical division cycle, transitioning into the NE as it divides at mitosis. On the basis of these findings, we propose that DdCenB is required at interphase to maintain proper nuclear architecture, and before delocalizing from the nucleus, DdCenB is part of the centrosome duplication machinery. PMID:19465563

  5. Assessing the role of the ASP56/CAP homologue of Dictyostelium discoideum and the requirements for subcellular localization.

    PubMed

    Noegel, A A; Rivero, F; Albrecht, R; Janssen, K P; Köhler, J; Parent, C A; Schleicher, M

    1999-10-01

    The CAP (cyclase-associated protein) homologue of Dictyostelium discoideum is a phosphatidylinositol 4,5-bisphosphate (PIP(2)) regulated G-actin sequestering protein which is present in the cytosol and shows enrichment at plasma membrane regions. It is composed of two domains separated by a proline rich stretch. The sequestering activity has been localized to the C-terminal domain of the protein, whereas the presence of the N-terminal domain seems to be required for PIP(2)-regulation of the sequestering activity. Here we have constructed GFP-fusions of N- and C-domain and found that the N-terminal domain showed CAP-specific enrichment at the anterior and posterior ends of cells like endogenous CAP irrespective of the presence of the proline rich region. Mutant cells expressing strongly reduced levels of CAP were generated by homologous recombination. They had an altered cell morphology with very heterogeneous cell sizes and exhibited a cytokinesis defect. Growth on bacteria was normal both in suspension and on agar plates as was phagocytosis of yeast and bacteria. In suspension in axenic medium mutant cells grew more slowly and did not reach saturation densities observed for wild-type cells. This was paralleled by a reduction in fluid phase endocytosis. Development was delayed by several hours under all conditions assayed, furthermore, motile behaviour was affected.

  6. Dictyostelium cells migrate similarly on surfaces of varying chemical composition.

    PubMed

    McCann, Colin P; Rericha, Erin C; Wang, Chenlu; Losert, Wolfgang; Parent, Carole A

    2014-01-01

    During cell migration, cell-substrate binding is required for pseudopod anchoring to move the cell forward, yet the interactions with the substrate must be sufficiently weak to allow parts of the cell to de-adhere in a controlled manner during typical protrusion/retraction cycles. Mammalian cells actively control cell-substrate binding and respond to extracellular conditions with localized integrin-containing focal adhesions mediating mechanotransduction. We asked whether mechanotransduction also occurs during non-integrin mediated migration by examining the motion of the social amoeba Dictyostelium discoideum, which is thought to bind non-specifically to surfaces. We discovered that Dictyostelium cells are able to regulate forces generated by the actomyosin cortex to maintain optimal cell-surface contact area and adhesion on surfaces of various chemical composition and that individual cells migrate with similar speed and contact area on the different surfaces. In contrast, during collective migration, as observed in wound healing and metastasis, the balance between surface forces and protrusive forces is altered. We found that Dictyostelium collective migration dynamics are strongly affected when cells are plated on different surfaces. These results suggest that the presence of cell-cell contacts, which appear as Dictyostelium cells enter development, alter the mechanism cells use to migrate on surfaces of varying composition.

  7. Dictyostelium discoideum Dgat2 can substitute for the essential function of Dgat1 in triglyceride production but not in ether lipid synthesis.

    PubMed

    Du, Xiaoli; Herrfurth, Cornelia; Gottlieb, Thomas; Kawelke, Steffen; Feussner, Kristin; Rühling, Harald; Feussner, Ivo; Maniak, Markus

    2014-04-01

    Triacylglycerol (TAG), the common energy storage molecule, is formed from diacylglycerol and a coenzyme A-activated fatty acid by the action of an acyl coenzyme A:diacylglycerol acyltransferase (DGAT). In order to conduct this step, most organisms rely on more than one enzyme. The two main candidates in Dictyostelium discoideum are Dgat1 and Dgat2. We show, by creating single and double knockout mutants, that the endoplasmic reticulum (ER)-localized Dgat1 enzyme provides the predominant activity, whereas the lipid droplet constituent Dgat2 contributes less activity. This situation may be opposite from what is seen in mammalian cells. Dictyostelium Dgat2 is specialized for the synthesis of TAG, as is the mammalian enzyme. In contrast, mammalian DGAT1 is more promiscuous regarding its substrates, producing diacylglycerol, retinyl esters, and waxes in addition to TAG. The Dictyostelium Dgat1, however, produces TAG, wax esters, and, most interestingly, also neutral ether lipids, which represent a significant constituent of lipid droplets. Ether lipids had also been found in mammalian lipid droplets, but the role of DGAT1 in their synthesis was unknown. The ability to form TAG through either Dgat1 or Dgat2 activity is essential for Dictyostelium to grow on bacteria, its natural food substrate.

  8. Studies of the cAMP mediated aggregation in Dictyostelium discoideum: receptor mediated activation of the adenylate cyclase

    SciTech Connect

    Theibert, W.E.A.B.

    1985-01-01

    Dictyostelium discoideum, a eukaryotic amoeba of the cellular slime mold family, provides an interesting paradigm in developmental biology. During development, hundreds of thousands of cells aggregate to form a multicellular aggregate. Aggregation is mediated by chemotaxis and chemical signaling. Waves of adenosine 3'-5' cyclic monophosphate (cAMP) propagate through the monolayer and provide transient gradients for chemotaxis. The author has used a reversible inhibitor of the cAMP signaling response to demonstrate that adaptation to cAMP is independent of the activation of the adenylate cyclase and therefore is not caused by the rise in intracellular cAMP. Next, it is shown that adenosine inhibits the cAMP signaling response. Inhibition is rapid, reversible, and depends on the cAMP stimulus concentration. Then the specificity of the cAMP receptors which mediates signaling is determined and compared with the receptors which mediate chemotaxis, the cGMP response, and cAMP binding antagonism. The cAMP surface receptor has been identified by photoaffinity labeling intact cells with (/sup 32/P)-8-N/sub 3/-cAMP using an ammonium sulfate binding stabilization technique. The photoactivated ligand specifically labels a polypeptide, localized to the membrane fraction, which migrates as a closely spaced doublet on SDS Page.

  9. Absence of catalytic domain in a putative protein kinase C (PkcA) suppresses tip dominance in Dictyostelium discoideum.

    PubMed

    Mohamed, Wasima; Ray, Sibnath; Brazill, Derrick; Baskar, Ramamurthy

    2015-09-01

    A number of organisms possess several isoforms of protein kinase C but little is known about the significance of any specific isoform during embryogenesis and development. To address this we characterized a PKC ortholog (PkcA; DDB_G0288147) in Dictyostelium discoideum. pkcA expression switches from prestalk in mound to prespore in slug, indicating a dynamic expression pattern. Mutants lacking the catalytic domain of PkcA (pkcA(-)) did not exhibit tip dominance. A striking phenotype of pkcA- was the formation of an aggregate with a central hollow, and aggregates later fragmented to form small mounds, each becoming a fruiting body. Optical density wave patterns of cAMP in the late aggregates showed several cAMP wave generation centers. We attribute these defects in pkcA(-) to impaired cAMP signaling, altered cell motility and decreased expression of the cell adhesion molecules - CadA and CsaA. pkcA(-) slugs showed ectopic expression of ecmA in the prespore region. Further, the use of a PKC-specific inhibitor, GF109203X that inhibits the activity of catalytic domain phenocopied pkcA(-).

  10. Absence of catalytic domain in a putative protein kinase C (PkcA) suppresses tip dominance in Dictyostelium discoideum

    PubMed Central

    Mohamed, Wasima; Ray, Sibnath; Brazill, Derrick; Baskar, Ramamurthy

    2017-01-01

    A number of organisms possess several isoforms of protein kinase C but little is known about the significance of any specific isoform during embryogenesis and development. To address this we characterized a PKC ortholog (PkcA; DDB_G0288147) in Dictyostelium discoideum. pkcA expression switches from prestalk in mound to prespore in slug, indicating a dynamic expression pattern. Mutants lacking the catalytic domain of PkcA (pkcA−) did not exhibit tip dominance. A striking phenotype of pkcA− was the formation of an aggregate with a central hollow, and aggregates later fragmented to form small mounds, each becoming a fruiting body. Optical density wave patterns of cAMP in the late aggregates showed several cAMP wave generation centers. We attribute these defects in pkcA− to impaired cAMP signaling, altered cell motility and decreased expression of the cell adhesion molecules – CadA and CsaA. pkcA− slugs showed ectopic expression of ecmA in the prespore region. Further, the use of a PKC-specific inhibitor, GF109203X that inhibits the activity of catalytic domain phenocopied pkcA−. PMID:26183108

  11. Properties of the Kinesin-1 motor DdKif3 from Dictyostelium discoideum.

    PubMed

    Röhlk, Christian; Rohlfs, Meino; Leier, Sven; Schliwa, Manfred; Liu, Xiao; Parsch, John; Woehlke, Günther

    2008-04-01

    The amoeba Dictyostelium discoideum possesses genes for 13 different kinesins. Here we characterize DdKif3, a member of the Kinesin-1 family. Kinesin-1 motors form homodimers that can move micrometer-long distances on microtubules using the energy derived from ATP hydrolysis. We expressed recombinant motors in Escherichia coli and tested them in different in vitro assays. Full-length and truncated Kif3 motors were active in gliding and ATPase assays. They showed a strong dependence on ionic strength. Like the full-length motor, the truncated DdKif3-592 motor (aa 1-592; comprising motor domain, neck, and partial stalk) reached its maximum speed of around 2.0micrcom s(-1) at a potassium acetate concentration of 200mM. The shortened DdKif3-342 motor (aa 1-342; comprising motor domain, partial neck) showed a high ATP turnover, comparable to that of the fungal Kinesin-1, Nkin. Results from the duty cycle calculations and gliding assays indicate that DdKif3 is a processive motor. A GFP-fusion protein revealed a mainly cytoplasmic localization of DdKif3. Immunofluorescence staining makes an association with the endoplasmic reticulum or mitochondria unlikely. Despite a similar phylogenetic distance to both metazoa and fungi, in terms of its biochemical properties DdKif3 revealed a closer similarity to fungal than animal kinesins.

  12. Micrometer-Scale Membrane Transition of Supported Lipid Bilayer Membrane Reconstituted with Cytosol of Dictyostelium discoideum

    PubMed Central

    Takahashi, Kei; Toyota, Taro

    2017-01-01

    Background: The transformation of the supported lipid bilayer (SLB) membrane by extracted cytosol from living resources, has recently drawn much attention. It enables us to address the question of whether the purified phospholipid SLB membrane, including lipids related to amoeba locomotion, which was discussed in many previous studies, exhibits membrane deformation in the presence of cytosol extracted from amoeba; Methods: In this report, a method for reconstituting a supported lipid bilayer (SLB) membrane, composed of purified phospholipids and cytosol extracted from Dictyostelium discoideum, is described. This technique is a new reconstitution method combining the artificial constitution of membranes with the reconstitution using animate cytosol (without precise purification at a molecular level), contributing to membrane deformation analysis; Results: The morphology transition of a SLB membrane composed of phosphatidylcholines, after the addition of cytosolic extract, was traced using a confocal laser scanning fluorescence microscope. As a result, pore formation in the SLB membrane was observed and phosphatidylinositides incorporated into the SLB membrane tended to suppress pore formation and expansion; Conclusions: The current findings imply that phosphatidylinositides have the potential to control cytoplasm activity and bind to a phosphoinositide-containing SLB membrane. PMID:28272354

  13. Microtubules Are Essential for Mitochondrial Dynamics-Fission, Fusion, and Motility-in Dictyostelium discoideum.

    PubMed

    Woods, Laken C; Berbusse, Gregory W; Naylor, Kari

    2016-01-01

    Mitochondrial function is dependent upon mitochondrial structure which is in turn dependent upon mitochondrial dynamics, including fission, fusion, and motility. Here we examined the relationship between mitochondrial dynamics and the cytoskeleton in Dictyostelium discoideum. Using time-lapse analysis, we quantified mitochondrial fission, fusion, and motility in the presence of cytoskeleton disrupting pharmaceuticals and the absence of the potential mitochondria-cytoskeleton linker protein, CluA. Our results indicate that microtubules are essential for mitochondrial movement, as well as fission and fusion; actin plays a less significant role, perhaps selecting the mitochondria for transport. We also suggest that CluA is not a linker protein but plays an unidentified role in mitochondrial fission and fusion. The significance of our work is to gain further insight into the role the cytoskeleton plays in mitochondrial dynamics and function. By better understanding these processes we can better appreciate the underlying mitochondrial contributions to many neurological disorders characterized by altered mitochondrial dynamics, structure, and/or function.

  14. Microtubules Are Essential for Mitochondrial Dynamics–Fission, Fusion, and Motility–in Dictyostelium discoideum

    PubMed Central

    Woods, Laken C.; Berbusse, Gregory W.; Naylor, Kari

    2016-01-01

    Mitochondrial function is dependent upon mitochondrial structure which is in turn dependent upon mitochondrial dynamics, including fission, fusion, and motility. Here we examined the relationship between mitochondrial dynamics and the cytoskeleton in Dictyostelium discoideum. Using time-lapse analysis, we quantified mitochondrial fission, fusion, and motility in the presence of cytoskeleton disrupting pharmaceuticals and the absence of the potential mitochondria-cytoskeleton linker protein, CluA. Our results indicate that microtubules are essential for mitochondrial movement, as well as fission and fusion; actin plays a less significant role, perhaps selecting the mitochondria for transport. We also suggest that CluA is not a linker protein but plays an unidentified role in mitochondrial fission and fusion. The significance of our work is to gain further insight into the role the cytoskeleton plays in mitochondrial dynamics and function. By better understanding these processes we can better appreciate the underlying mitochondrial contributions to many neurological disorders characterized by altered mitochondrial dynamics, structure, and/or function. PMID:27047941

  15. Identification of Proteins Associated with Multilamellar Bodies Produced by Dictyostelium discoideum

    PubMed Central

    Denoncourt, Alix M.; Paquet, Valérie E.; Sedighi, Ahmadreza; Charette, Steve J.

    2016-01-01

    Dictyostelium discoideum amoebae produce and secrete multilamellar bodies (MLBs) when fed digestible bacteria. The aim of the present study was to elucidate the proteic content of MLBs. The lipid composition of MLBs is mainly amoebal in origin, suggesting that MLB formation is a protozoa-driven process that could play a significant role in amoebal physiology. We identified four major proteins on purified MLBs using mass spectrometry in order to better understand the molecular mechanisms governing MLB formation and, eventually, to elucidate the true function of MLBs. These proteins were SctA, PhoPQ, PonC and a protein containing a cytidine/deoxycytidylate deaminase (CDD) zinc-binding region. SctA is a component of pycnosomes, which are membranous materials that are continuously secreted by amoebae. The presence of SctA on MLBs was confirmed by immunofluorescence and Western blotting using a specific anti-SctA antibody. The CDD protein may be one of the proteins recognized by the H36 antibody, which was used as a MLB marker in a previous study. The function of the CDD protein is unknown. Immunofluorescence and flow cytometric analyses confirmed that the H36 antibody is a better marker of MLBs than the anti-SctA antibody. This study is an additional step to elucidate the potential role of MLBs and revealed that only a small set of proteins appeared to be present on MLBs. PMID:27340834

  16. The rate and effects of spontaneous mutation on fitness traits in the social amoeba, Dictyostelium discoideum.

    PubMed

    Hall, David W; Fox, Sara; Kuzdzal-Fick, Jennie J; Strassmann, Joan E; Queller, David C

    2013-07-08

    We performed a mutation accumulation (MA) experiment in the social amoeba Dictyostelium discoideum to estimate the rate and distribution of effects of spontaneous mutations affecting eight putative fitness traits. We found that the per-generation mutation rate for most fitness components is 0.0019 mutations per haploid genome per generation or larger. This rate is an order of magnitude higher than estimates for fitness components in the unicellular eukaryote Saccharomyces cerevisiae, even though the base-pair substitution rate is two orders of magnitude lower. The high rate of fitness-altering mutations observed in this species may be partially explained by a large mutational target relative to S. cerevisiae. Fitness-altering mutations also may occur primarily at simple sequence repeats, which are common throughout the genome, including in coding regions, and may represent a target that is particularly likely to give fitness effects upon mutation. The majority of mutations had deleterious effects on fitness, but there was evidence for a substantial fraction, up to 40%, being beneficial for some of the putative fitness traits. Competitive ability within the multicellular slug appears to be under weak directional selection, perhaps reflecting the fact that slugs are sometimes, but not often, comprised of multiple clones in nature. Evidence for pleiotropy among fitness components across MA lines was absent, suggesting that mutations tend to act on single fitness components.

  17. Actin-Interacting Protein 1 Contributes to Intranuclear Rod Assembly in Dictyostelium discoideum

    PubMed Central

    Ishikawa-Ankerhold, Hellen C.; Daszkiewicz, Wioleta; Schleicher, Michael; Müller-Taubenberger, Annette

    2017-01-01

    Intranuclear rods are aggregates consisting of actin and cofilin that are formed in the nucleus in consequence of chemical or mechanical stress conditions. The formation of rods is implicated in a variety of pathological conditions, such as certain myopathies and some neurological disorders. It is still not well understood what exactly triggers the formation of intranuclear rods, whether other proteins are involved, and what the underlying mechanisms of rod assembly or disassembly are. In this study, Dictyostelium discoideum was used to examine appearance, stages of assembly, composition, stability, and dismantling of rods. Our data show that intranuclear rods, in addition to actin and cofilin, are composed of a distinct set of other proteins comprising actin-interacting protein 1 (Aip1), coronin (CorA), filactin (Fia), and the 34 kDa actin-bundling protein B (AbpB). A finely tuned spatio-temporal pattern of protein recruitment was found during formation of rods. Aip1 is important for the final state of rod compaction indicating that Aip1 plays a major role in shaping the intranuclear rods. In the absence of both Aip1 and CorA, rods are not formed in the nucleus, suggesting that a sufficient supply of monomeric actin is a prerequisite for rod formation. PMID:28074884

  18. Differentiation-inducing factor 2 modulates chemotaxis via the histidine kinase DhkC-dependent pathway in Dictyostelium discoideum.

    PubMed

    Kuwayama, Hidekazu; Kubohara, Yuzuru

    2016-03-01

    Differentiation-inducing factor 1(DIF-1) and DIF-2 are signaling molecules that control chemotaxis in Dictyostelium discoideum. Whereas DIF-1 suppresses chemotaxis in shallow cAMP gradients, DIF-2 enhances chemotaxis under the same conditions via a phosphodiesterase, response regulator A (RegA), which is a part of the DhkC-RdeA-RegA two-component signaling system. In this study, to investigate the mechanism of the chemotaxis regulation by DIF-2, we examined the effects of DIF-2 (and DIF-1) on chemotaxis in rdeA(-) and dhkC(-) mutant strains. In the parental wild-type strains, chemotactic cell movement was suppressed with DIF-1 and enhanced with DIF-2 in shallow cAMP gradients. In contrast, in both rdeA(-) and dhkC(-) strains, chemotaxis was suppressed with DIF-1 but unaffected by DIF-2. The results suggest that DIF-2 modulates chemotaxis via the DhkC-RdeA-RegA signaling system.

  19. mRNA stabilization controls the expression of a class of developmentally regulated genes in Dictyostelium discoideum

    PubMed Central

    Mangiarotti, Giorgio; Giorda, Roberto; Ceccarelli, Adriano; Perlo, Carla

    1985-01-01

    During the development of Dictyostelium discoideum, several thousand new mRNA species appear in the cytoplasm after the cells have formed stable aggregates. Here we show that six of these late mRNAs, corresponding to six clones randomly chosen from a genomic library, are synthesized from the very beginning of development at a rate comparable to that observed late in development but that transcripts do not accumulate until after aggregation. The early- and late-synthesized mRNAs are identical in size and compete with each other for hybridization to the genomic clones. The early-synthesized mRNAs do not accumulate in the cytoplasm in the preaggregation stage because they are very unstable. Their stability, estimated from the kinetics of incorporation during continuous labeling with 32P, increases by perhaps an order of magnitude in the postaggregation stage. We conclude that mRNA stabilization is the major controlling factor of the expression of these genes. Images PMID:16593597

  20. Probabilistic transition from unstable predator-prey interaction to stable coexistence of Dictyostelium discoideum and Escherichia coli.

    PubMed

    Kihara, Kumiko; Mori, Kotaro; Suzuki, Shingo; Hosoda, Kazufumi; Yamada, Akito; Matsuyama, Shin-ichi; Kashiwagi, Akiko; Yomo, Tetsuya

    2011-03-01

    Predator-prey interactions have been found at all levels within ecosystems. Despite their ecological ubiquity and importance, the process of transition to a stable coexistent state has been poorly verified experimentally. To investigate the stabilization process of predator-prey interactions, we previously constructed a reproducible experimental predator-prey system between Dictyostelium discoideum and Escherichia coli, and showed that the phenotypically changed E. coli contributed to stabilization of the system. In the present study, we focused on the transition to stable coexistence of both species after the phenotypic change in E. coli. Analysis of E. coli cells isolated from co-culture plates as single colony enabled us to readily identify the appearance of phenotypically changed E. coli that differed in colony morphology and growth rate. It was also demonstrated that two types of viscous colony, i.e., the dense-type and sparse-type, differing in spatial distribution of both species emerged probabilistically and all of the viscous colonies maintained stably were of the sparse-type. These results suggest that the phenotypically changed E. coli may produce two types of viscous colonies probabilistically. The difference in spatial distribution would affect localized interactions between both species and then cause probabilistic stabilization of predator-prey interactions.

  1. Genetic Diversity in Cellular Slime Molds: Allozyme Electrophoresis and a Monoclonal Antibody Reveal Cryptic Species among Dictyostelium discoideum Strains

    PubMed Central

    Briscoe, David A.; Gooley, Andrew A.; Bernstein, R. L.; McKay, George M.; Williams, Keith L.

    1987-01-01

    Cellular slime molds have been classified on the basis of a small number of descriptive criteria such as fruiting body color and morphology, and, in heterothallic species, by assignment to compatible mating groups. However, some isolates which are morphologically classified as conspecific do not fall into a simple mating-type classification; for example some are asexual or homothallic. An increasing interest in inter-strain genetic variation in studies of development and simple behavior has led us to reassess genetic relationships among a number of frequently used isolates. Allozyme electrophoresis of 16 soluble enzymes and use of a monoclonal antibody show that there is relatively little genetic diversity among sexually competent Dictyostelium discoideum isolates, despite considerable variation in geographic origin and time since isolation in the laboratory. In contrast a pair of asexual strains and each of two homothallic strains are genetically quite distinct and differ sufficiently from each other, and from sexually competent isolates, to warrant their recognition as separate species. There are probably four biological species represented in the supposedly D. discoideum isolates studied. This heterogeneity extends to other cellular slime mold species. Each of three isolates of Dictyostelium purpureum is genetically distinct from the others. Limited analysis of other cellular slime molds indicates that the generic distinction of Dictyostelium and Polysphondylium must be questioned. This study emphasizes that caution should be applied in classifying simple organisms on morphological criteria. PMID:17246401

  2. Cytosolic acidification as a signal mediating hyperosmotic stress responses in Dictyostelium discoideum

    PubMed Central

    Pintsch, Tanja; Satre, Michel; Klein, Gérard; Martin, Jean-Baptiste; Schuster, Stephan C

    2001-01-01

    Background Dictyostelium cells exhibit an unusual response to hyperosmolarity that is distinct from the response in other organisms investigated: instead of accumulating compatible osmolytes as it has been described for a wide range of organisms, Dictyostelium cells rearrange their cytoskeleton and thereby build up a rigid network which is believed to constitute the major osmoprotective mechanism in this organism. To gain more insight into the osmoregulation of this amoeba, we investigated physiological processes affected under hyperosmotic conditions in Dictyostelium. Results We determined pH changes in response to hyperosmotic stress using FACS or 31P-NMR. Hyperosmolarity was found to acidify the cytosol from pH 7.5 to 6.8 within 5 minutes, whereas the pH of the endo-lysosomal compartment remained constant. Fluid-phase endocytosis was identified as a possible target of cytosolic acidification, as the inhibition of endocytosis observed under hypertonic conditions can be fully attributed to cytosolic acidification. In addition, a deceleration of vesicle mobility and a decrease in the NTP pool was observed. Conclusion Together, these results indicate that hyperosmotic stress triggers pleiotropic effects, which are partially mediated by a pH signal and which all contribute to the downregulation of cellular activity. The comparison of our results with the effect of hyperosmolarity and intracellular acidification on receptor-mediated endocytosis in mammalian cells reveals striking similarities, suggesting the hypothesis of the same mechanism of inhibition by low internal pH. PMID:11415467

  3. A membrane cytoskeleton from Dictyostelium discoideum. I. Identification and partial characterization of an actin-binding activity

    PubMed Central

    1981-01-01

    Dictyostelium discoideum plasma membranes isolated by each of three procedures bind F-actin. The interactions between these membranes and actin are examined by a novel application of falling ball viscometry. Treating the membranes as multivalent actin-binding particles analogous to divalent actin-gelation factors, we observe large increases in viscosity (actin cross-linking) when membranes of depleted actin and myosin are incubated with rabbit skeletal muscle F-actin. Pre- extraction of peripheral membrane proteins with chaotropes or the inclusion of Triton X-100 during the assay does not appreciably diminish this actin cross-linking activity. Lipid vesicles, heat- denatured membranes, proteolyzed membranes, or membranes containing endogenous actin show minimal actin cross-linking activity. Heat- denatured, but not proteolyzed, membranes regain activity when assayed in the presence of Triton X-100. Thus, integral membrane proteins appear to be responsible for some or all of the actin cross-linking activity of D. discoideum membranes. In the absence of MgATP, Triton X- 100 extraction of isolated D. discoideum membranes results in a Triton- insoluble residue composed of actin, myosin, and associated membrane proteins. The inclusion of MgATP before and during Triton extraction greatly diminishes the amount of protein in the Triton-insoluble residue without appreciably altering its composition. Our results suggest the existence of a protein complex stabilized by actin and/or myosin (membrane cytoskeleton) associated with the D. discoideum plasma membrane. PMID:6894148

  4. Characterization of a 1,4-{beta}-D-glucan synthase from Dictyostelium discoideum. Progress report, May 1990--January 1992

    SciTech Connect

    Blanton, R.L.

    1992-01-15

    Various aspects of research concerning Dictyostelium discoideum are presented. The initial focus of this project was upon: the characterization of potential probes for the cellulose synthase (antibody and nucleic acid), the determination of the cultural induction conditions of cellulose synthesis, the solubilization of the enzyme activity, the development of a non-inhibitory disruption buffer, the generation and isolation of mutant strains deficient in cellulose synthesis, and the development of the capability to determine the degree of polymerization of the in vitro product. I have briefly summarized our most significant findings with only selected data sets being shown in this report in the interest of brevity.

  5. mRNA decay rates in late-developing Dictyostelium discoideum cells are heterogeneous, and cyclic AMP does not act directly to stabilize cell-type-specific mRNAs.

    PubMed Central

    Manrow, R E; Jacobson, A

    1988-01-01

    We reevaluated the use of 32PO4 pulse-chases for analyzing mRNA decay rates in late-developing Dictyostelium cells. We found that completely effective PO4 chases could not be obtained in developing cells and that, as a consequence, the decay rates exhibited by some mRNAs were influenced by the rates at which they were transcribed. In developing cells disaggregated in the presence of cyclic AMP, the poly(A)+ mRNA population turned over with an apparent half-life of 4 h, individual mRNA decay rates were heterogeneous, and some prestalk and prespore mRNAs appeared to decay with biphasic kinetics. In cells disaggregated in the absence of cyclic AMP, all prestalk and prespore mRNAs decayed with biphasic kinetics. During the first 1 to 1.5 h after disaggregation in the absence of cyclic AMP, the cell-type-specific mRNAs were selectively degraded, decaying with half-lives of 20 to 30 min; thereafter, the residual prestalk and prespore mRNA molecules decayed at rates that were similar to those measured in the presence of cyclic AMP. This short-term labilization of cell-type-specific mRNAs was observed even for those species not requiring cyclic AMP for their accumulation in developing cells. The observation that cell-type specific mRNAs can decay at similar rates in disaggregated cells with or without cyclic AMP indicates that this compound does not act directly to stabilize prestalk and prespore mRNAs during development and that its primary role in the maintenance of cyclic-AMP-dependent mRNAs is likely to be transcriptional. Images PMID:2847029

  6. Extracellular calmodulin regulates growth and cAMP-mediated chemotaxis in Dictyostelium discoideum

    SciTech Connect

    O'Day, Danton H.; Huber, Robert J.; Suarez, Andres

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer Extracellular calmodulin is present throughout growth and development in Dictyostelium. Black-Right-Pointing-Pointer Extracellular calmodulin localizes within the ECM during development. Black-Right-Pointing-Pointer Extracellular calmodulin inhibits cell proliferation and increases chemotaxis. Black-Right-Pointing-Pointer Extracellular calmodulin exists in eukaryotic microbes. Black-Right-Pointing-Pointer Extracellular calmodulin may be functionally as important as intracellular calmodulin. -- Abstract: The existence of extracellular calmodulin (CaM) has had a long and controversial history. CaM is a ubiquitous calcium-binding protein that has been found in every eukaryotic cell system. Calcium-free apo-CaM and Ca{sup 2+}/CaM exert their effects by binding to and regulating the activity of CaM-binding proteins (CaMBPs). Most of the research done to date on CaM and its CaMBPs has focused on their intracellular functions. The presence of extracellular CaM is well established in a number of plants where it functions in proliferation, cell wall regeneration, gene regulation and germination. While CaM has been detected extracellularly in several animal species, including frog, rat, rabbit and human, its extracellular localization and functions are less well established. In contrast the study of extracellular CaM in eukaryotic microbes remains to be done. Here we show that CaM is constitutively expressed and secreted throughout asexual development in Dictyostelium where the presence of extracellular CaM dose-dependently inhibits cell proliferation but increases cAMP mediated chemotaxis. During development, extracellular CaM localizes within the slime sheath where it coexists with at least one CaMBP, the matricellular CaM-binding protein CyrA. Coupled with previous research, this work provides direct evidence for the existence of extracellular CaM in the Dictyostelium and provides insight into its functions in this model amoebozoan.

  7. Reversible inhibition of movement in the amoebae Dictyostelium discoideum and its effect on chemoattractant recognition.

    PubMed

    Waligórska, Agnieszka; Wianecka-Skoczeń, Magdalena; Korohoda, Włodzimierz

    2008-01-01

    The cell fixatives formaldehyde and KMnO4 at low concentrations reversibly inhibit the movement of D. discoideum amoebae without directly interfering with cell viability. This inhibition of cell movement is accompanied by the decreased attachment of cells to substratum. When the tenacity and attachment of immobilized cells are artificially increased by compressing cells between two glass surfaces, the amoebae begin to move even in the presence of the fixatives. Amoebae starved for 24 hours, subjected to fixatives and a mineral salt solution in which they remained motionless, maintained chemotactic responses to folic acid and only after a few hours of active locomotion became reactive to cAMP, in contrast to amoebae that reacted to cAMP after starvation in the absence of fixatives.

  8. Excitable waves and direction-sensing in Dictyostelium discoideum: steps towards a chemotaxis model

    NASA Astrophysics Data System (ADS)

    Bhowmik, Arpan; Rappel, Wouter-Jan; Levine, Herbert

    2016-02-01

    In recent years, there have been significant advances in our understanding of the mechanisms underlying chemically directed motility by eukaryotic cells such as Dictyostelium. In particular, the local excitation and global inhibition (LEGI) model has proven capable of providing a framework for quantitatively explaining many experiments that present Dictyostelium cells with tailored chemical stimuli and monitor their subsequent polarization. In their natural setting, cells generate their own directional signals via the detection and secretion of cyclic adenosine monophosphate (cAMP). Here, we couple the LEGI approach to an excitable medium model of the cAMP wave-field that is propagated by the cells and investigate the possibility for this class of models to enable accurate chemotaxis to the cAMP waveforms expected in vivo. Our results indicate that the ultra-sensitive version of the model does an excellent job in providing natural wave rectification, thereby providing a compelling solution to the ‘back-of-the-wave paradox’ during cellular aggregation.

  9. Lack of Ecological and Life History Context Can Create the Illusion of Social Interactions in Dictyostelium discoideum.

    PubMed

    Martínez-García, Ricardo; Tarnita, Corina E

    2016-12-01

    Studies of social microbes often focus on one fitness component (reproductive success within the social complex), with little information about or attention to other stages of the life cycle or the ecological context. This can lead to paradoxical results. The life cycle of the social amoeba Dictyostelium discoideum includes a multicellular stage in which not necessarily clonal amoebae aggregate upon starvation to form a possibly chimeric (genetically heterogeneous) fruiting body made of dead stalk cells and spores. The lab-measured reproductive skew in the spores of chimeras indicates strong social antagonism that should result in low genotypic diversity, which is inconsistent with observations from nature. Two studies have suggested that this inconsistency stems from the one-dimensional assessment of fitness (spore production) and that the solution lies in tradeoffs between multiple life-history traits, e.g.: spore size versus viability; and spore-formation (via aggregation) versus staying vegetative (as non-aggregated cells). We develop an ecologically-grounded, socially-neutral model (i.e. no social interactions between genotypes) for the life cycle of social amoebae in which we theoretically explore multiple non-social life-history traits, tradeoffs and tradeoff-implementing mechanisms. We find that spore production comes at the expense of time to complete aggregation, and, depending on the experimental setup, spore size and viability. Furthermore, experimental results regarding apparent social interactions within chimeric mixes can be qualitatively recapitulated under this neutral hypothesis, without needing to invoke social interactions. This allows for simple potential resolutions to the previously paradoxical results. We conclude that the complexities of life histories, including social behavior and multicellularity, can only be understood in the appropriate multidimensional ecological context, when considering all stages of the life cycle.

  10. Lack of Ecological and Life History Context Can Create the Illusion of Social Interactions in Dictyostelium discoideum

    PubMed Central

    Martínez-García, Ricardo; Tarnita, Corina E.

    2016-01-01

    Studies of social microbes often focus on one fitness component (reproductive success within the social complex), with little information about or attention to other stages of the life cycle or the ecological context. This can lead to paradoxical results. The life cycle of the social amoeba Dictyostelium discoideum includes a multicellular stage in which not necessarily clonal amoebae aggregate upon starvation to form a possibly chimeric (genetically heterogeneous) fruiting body made of dead stalk cells and spores. The lab-measured reproductive skew in the spores of chimeras indicates strong social antagonism that should result in low genotypic diversity, which is inconsistent with observations from nature. Two studies have suggested that this inconsistency stems from the one-dimensional assessment of fitness (spore production) and that the solution lies in tradeoffs between multiple life-history traits, e.g.: spore size versus viability; and spore-formation (via aggregation) versus staying vegetative (as non-aggregated cells). We develop an ecologically-grounded, socially-neutral model (i.e. no social interactions between genotypes) for the life cycle of social amoebae in which we theoretically explore multiple non-social life-history traits, tradeoffs and tradeoff-implementing mechanisms. We find that spore production comes at the expense of time to complete aggregation, and, depending on the experimental setup, spore size and viability. Furthermore, experimental results regarding apparent social interactions within chimeric mixes can be qualitatively recapitulated under this neutral hypothesis, without needing to invoke social interactions. This allows for simple potential resolutions to the previously paradoxical results. We conclude that the complexities of life histories, including social behavior and multicellularity, can only be understood in the appropriate multidimensional ecological context, when considering all stages of the life cycle. PMID:27977666

  11. Structure of DRE, a retrotransposable element which integrates with position specificity upstream of Dictyostelium discoideum tRNA genes.

    PubMed Central

    Marschalek, R; Hofmann, J; Schumann, G; Gösseringer, R; Dingermann, T

    1992-01-01

    Different Dictyostelium discoideum strains contain between 2 and 200 copies of a retrotransposable element termed DRE (Dictyostelium repetitive element). From the analysis of more than 50 elements, it can be concluded that DRE elements always occur 50 +/- 3 nucleotides upstream of tRNA genes. All analyzed clones contain DRE in a constant orientation relative to the tRNA gene, implying orientation specificity as well as position specificity. DRE contains two open reading frames which are flanked by nonidentical terminal repeats. Long terminal repeats (LTRs) are composed of three distinct modules, called A, B, and C. The tRNA gene-proximal LTR is characterized by one or multiple A modules followed by a single B module (AnB). With respect to the distal LTR, two different subforms of DRE have been isolated. The majority of isolated clones contains a distal LTR composed of a B module followed by a C module (BC), whereas the distal LTR of the other subform contains a consecutive array of a B module, a C module, a slightly altered A module, another B module, and another C module (BC.ABC). Full-length as well as smaller transcripts from DRE elements have been detected, but in comparison with the high copy number in D. discoideum strains derived from the wild-type strain NC4, transcription is rather poor. Images PMID:1309589

  12. Structure of the 34 kDa F-actin-bundling protein ABP34 from Dictyostelium discoideum.

    PubMed

    Kim, Min-Kyu; Kim, Ji-Hye; Kim, Ji-Sun; Kang, Sa-Ouk

    2015-09-01

    The crystal structure of the 34 kDa F-actin-bundling protein ABP34 from Dictyostelium discoideum was solved by Ca(2+)/S-SAD phasing and refined at 1.89 Å resolution. ABP34 is a calcium-regulated actin-binding protein that cross-links actin filaments into bundles. Its in vitro F-actin-binding and F-actin-bundling activities were confirmed by a co-sedimentation assay and transmission electron microscopy. The co-localization of ABP34 with actin in cells was also verified. ABP34 adopts a two-domain structure with an EF-hand-containing N-domain and an actin-binding C-domain, but has no reported overall structural homologues. The EF-hand is occupied by a calcium ion with a pentagonal bipyramidal coordination as in the canonical EF-hand. The C-domain structure resembles a three-helical bundle and superposes well onto the rod-shaped helical structures of some cytoskeletal proteins. Residues 216-244 in the C-domain form part of the strongest actin-binding sites (193-254) and exhibit a conserved sequence with the actin-binding region of α-actinin and ABP120. Furthermore, the second helical region of the C-domain is kinked by a proline break, offering a convex surface towards the solvent area which is implicated in actin binding. The F-actin-binding model suggests that ABP34 binds to the side of the actin filament and residues 216-244 fit into a pocket between actin subdomains -1 and -2 through hydrophobic interactions. These studies provide insights into the calcium coordination in the EF-hand and F-actin-binding site in the C-domain of ABP34, which are associated through interdomain interactions.

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

    PubMed

    Shimada, Nao; Nishio, Keiko; Maeda, Mineko; Urushihara, Hideko; Kawata, Takefumi

    2004-10-01

    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.

  14. Partial genetic suppression of a loss-of-function mutant of the neuronal ceroid lipofuscinosis-associated protease TPP1 in Dictyostelium discoideum.

    PubMed

    Phillips, Jonathan E; Gomer, Richard H

    2015-02-01

    Neuronal ceroid lipofuscinosis (NCL) is the most common childhood-onset neurodegenerative disease. NCL is inevitably fatal, and there is currently no treatment available. Children with NCL show a progressive decline in movement, vision and mental abilities, and an accumulation of autofluorescent deposits in neurons and other cell types. Late-infantile NCL is caused by mutations in the lysosomal protease tripeptidyl peptidase 1 (TPP1). TPP1 cleaves tripeptides from the N-terminus of proteins in vitro, but little is known about the physiological function of TPP1. TPP1 shows wide conservation in vertebrates but it is not found in Drosophila, Caenorhabditis elegans or Saccharomyces cerevisiae. Here, we characterize ddTpp1, a TPP1 ortholog present in the social amoeba Dictyostelium discoideum. Lysates from cells lacking ddTpp1 show a reduced but not abolished ability to cleave a TPP1 substrate, suggesting that other Dictyostelium enzymes can perform this cleavage. ddTpp1 and human TPP1 localize to the lysosome in Dictyostelium, indicating conserved function and trafficking. Cells that lack ddTpp1 show precocious multicellular development and a reduced ability to form spores during development. When cultured in autophagy-stimulating conditions, cells lacking ddTpp1 rapidly decrease in size and are less viable than wild-type cells, suggesting that one function of ddTpp1 could be to limit autophagy. Cells that lack ddTpp1 exhibit strongly impaired development in the presence of the lysosome-perturbing drug chloroquine, and this phenotype can be suppressed through a secondary mutation in the gene that we name suppressor of tpp1(-) A (stpA), which encodes a protein with some similarity to mammalian oxysterol-binding proteins (OSBPs). Taken together, these results suggest that targeting specific proteins could be a viable way to suppress the effects of loss of TPP1 function.

  15. Characterization of a third ras gene, rasB, that is expressed throughout the growth and development of Dictyostelium discoideum.

    PubMed

    Daniel, J; Spiegelman, G B; Weeks, G

    1993-04-01

    Previous reports have indicated that the cellular slime mold Dictyostelium discoideum possesses two ras genes (rasG and rasD) and one rap gene (rap1). All three genes are developmentally regulated, with each showing a different pattern of transcription during the Dictyostelium life cycle. To establish whether there are additional ras or rap genes in Dictyostelium, we used degenerate oligonucleotide primers to the highly conserved GTP-binding domains and both ras- and rap-unique sequences to amplify products from cDNA using the polymerase chain reaction (PCR). No additional rap genes were amplified, but a fragment whose nucleotide sequence predicted a novel ras gene was isolated. Using this PCR product as a probe, a full-length cDNA clone was isolated and sequenced. Its deduced amino acid sequence predicted a 197 amino acid protein that is 71% and 68% identical to RasG and RasD respectively. The new ras gene contains the conserved Ras-specific effector domain, the conserved binding site for the Ras-specific Y13-259 monoclonal antibody, and shows greater sequence similarity to the human H-Ras protein than to any other mammalian Ras protein. In view of this high level of identity to the ras gene subfamily, we have designated this gene rasB. Northern blot analysis has shown that rasB is developmentally regulated with maximum levels of a single 950-bp message detected during vegetative growth and the first 8 h of development.

  16. Analysis of chemotaxis in Dictyostelium

    PubMed Central

    Cai, Huaqing; Huang, Chuan-Hsiang; Devreotes, Peter N.; Iijima, Miho

    2012-01-01

    Dictyostelium discoideum is an excellent model organism for the study of directed cell migration since Dictyostelium cells show robust chemotactic responses to the chemoattractant cAMP. Many powerful experimental tools are applicable, including forward and reverse genetics, biochemistry, microscopy, and proteomics. Recent studies have demonstrated that many components involved in chemotaxis are functionally conserved between human neutrophils and Dictyostelium amoebae. In this section, we will describe how to define the functions of proteins that mediate and regulate cell motility, cell polarity, and directional sensing during chemotaxis in Dictyostelium. PMID:21909927

  17. Characterization and genetic mapping of modA. A mutation in the post-translational modification of the glycosidases of Dictyostelium discoideum.

    PubMed

    Free, S J; Schimke, R T; Freeze, H; Loomis, W F

    1978-06-25

    We have isolated a mutant of Dictyostelium discoideum, M31, which produces a reduced number of alpha-mannosidase-1 molecules per cell during the developmental program of the organism. We find that several of the glycosidases, a group of lysosomal proteins produced by D. discoideum, are altered in strain M31 and that this strain produces a reduced level of at least three of these activities. These enzymes do not share a common protein subunit but are known to share a common antigenic determinant which is, in part, carbohydrate in nature. In the wild type parent of M31, alpha-mannosidase-1 is modified by the addition of mannose and glucosamine (probably as N-acetylglucosamine) in the molar ratio of 5:2. alpha-Mannosidase-1 was also found to contain phosphoserine/phosphothreonine residues. alpha-Mannosidase-1 and other glycosidases are electrophoretically less negative when isolated from strain M31 than when isolated from wild type cells. The mutation present in M31, modA, appears to affect posttranslational modification, modA is a recessive mutation which we map onto linkage group I.

  18. Dictyostelium discoideum has a highly Q/N-rich proteome and shows an unusual resilience to protein aggregation.

    PubMed

    Malinovska, Liliana; Palm, Sandra; Gibson, Kimberley; Verbavatz, Jean-Marc; Alberti, Simon

    2015-05-19

    Many protein-misfolding diseases are caused by proteins carrying prion-like domains. These proteins show sequence similarity to yeast prion proteins, which can interconvert between an intrinsically disordered and an aggregated prion state. The natural presence of prions in yeast has provided important insight into disease mechanisms and cellular proteostasis. However, little is known about prions in other organisms, and it is not yet clear whether the findings in yeast can be generalized. Using bioinformatics tools, we show that Dictyostelium discoideum has the highest content of prion-like proteins of all organisms investigated to date, suggesting that its proteome has a high overall aggregation propensity. To study mechanisms regulating these proteins, we analyze the behavior of several well-characterized prion-like proteins, such as an expanded version of human huntingtin exon 1 (Q103) and the prion domain of the yeast prion protein Sup35 (NM), in D. discoideum. We find that these proteins remain soluble and are innocuous to D. discoideum, in contrast to other organisms, where they form cytotoxic cytosolic aggregates. However, when exposed to conditions that compromise molecular chaperones, these proteins aggregate and become cytotoxic. We show that the disaggregase Hsp101, a molecular chaperone of the Hsp100 family, dissolves heat-induced aggregates and promotes thermotolerance. Furthermore, prion-like proteins accumulate in the nucleus, where they are targeted by the ubiquitin-proteasome system. Our data suggest that D. discoideum has undergone specific adaptations that increase the proteostatic capacity of this organism and allow for an efficient regulation of its prion-like proteome.

  19. Dictyostelium discoideum has a highly Q/N-rich proteome and shows an unusual resilience to protein aggregation

    PubMed Central

    Malinovska, Liliana; Palm, Sandra; Gibson, Kimberley; Verbavatz, Jean-Marc; Alberti, Simon

    2015-01-01

    Many protein-misfolding diseases are caused by proteins carrying prion-like domains. These proteins show sequence similarity to yeast prion proteins, which can interconvert between an intrinsically disordered and an aggregated prion state. The natural presence of prions in yeast has provided important insight into disease mechanisms and cellular proteostasis. However, little is known about prions in other organisms, and it is not yet clear whether the findings in yeast can be generalized. Using bioinformatics tools, we show that Dictyostelium discoideum has the highest content of prion-like proteins of all organisms investigated to date, suggesting that its proteome has a high overall aggregation propensity. To study mechanisms regulating these proteins, we analyze the behavior of several well-characterized prion-like proteins, such as an expanded version of human huntingtin exon 1 (Q103) and the prion domain of the yeast prion protein Sup35 (NM), in D. discoideum. We find that these proteins remain soluble and are innocuous to D. discoideum, in contrast to other organisms, where they form cytotoxic cytosolic aggregates. However, when exposed to conditions that compromise molecular chaperones, these proteins aggregate and become cytotoxic. We show that the disaggregase Hsp101, a molecular chaperone of the Hsp100 family, dissolves heat-induced aggregates and promotes thermotolerance. Furthermore, prion-like proteins accumulate in the nucleus, where they are targeted by the ubiquitin–proteasome system. Our data suggest that D. discoideum has undergone specific adaptations that increase the proteostatic capacity of this organism and allow for an efficient regulation of its prion-like proteome. PMID:25941378

  20. Mutation of an EF-hand Ca(2+)-binding motif in phospholipase C of Dictyostelium discoideum: inhibition of activity but no effect on Ca(2+)-dependence.

    PubMed

    Drayer, A L; Meima, M E; Derks, M W; Tuik, R; van Haastert, P J

    1995-10-15

    Phosphoinositide-specific phospholipase C (PLC) is dependent on Ca2+ ions for substrate hydrolysis. The role of an EF-hand Ca(2+)-binding motif in Ca(2+)-dependent PLC activity was investigated by site-directed mutagenesis of the Dictyostelium discoideum PLC enzyme. Amino acid residues with oxygen-containing side chains at co-ordinates x, y, z, -x and -z of the putative Ca(2+)-binding-loop sequence were replaced by isoleucine (x), valine (y) or alanine (z, -x and -z). The mutated proteins were expressed in a Dictyostelium cell line with a disrupted plc gene displaying no endogenous PLC activity, and PLC activity was measured in cell lysates at different Ca2+ concentrations. Replacement of aspartate at position x, which is considered to play an essential role in Ca2+ binding, had little effect on Ca2+ affinity and maximal enzyme activity. A mutant with substitutions at both aspartate residues in position x and y also showed no decrease in Ca2+ affinity, whereas the maximal PLC activity was reduced by 60%. Introduction of additional mutations in the EF-hand revealed that the Ca2+ concentration giving half-maximal activity was unaltered, but PLC activity levels at saturating Ca2+ concentrations were markedly decreased. The results demonstrate that, although the EF-hand domain is required for enzyme activity, it is not the site that regulates the Ca(2+)-dependence of the PLC reaction.

  1. Flow-driven instabilities during aggregation and pattern formation of Dictyostelium Discoideum: Experiments and modeling

    NASA Astrophysics Data System (ADS)

    Gholami, Azam; Steinbock, Oliver; Zykov, Vladimir; Bodenschatz, Eberhard

    2013-03-01

    We report the first experimental verification of the Differential Flow Induced Chemical Instability (DIFICI) in a signaling chemotactic biological population, where a differential flow induces traveling waves in the signaling pattern. The traveling wave speed was observed to be proportional to the flow velocity while the wave period was 7 min, which is comparable to that of starved Dictyostelium cells. Analysis and numerical simulations of the Goldbeter model show that the resulting DIFICI wave patterns appear in the oscillatory regime. In the experiments, we observe that the DIFICI wave pattern disappears after 4-5 h of starvation. We extrapolated the Goldbeter model to the experimental situation. This suggests that the dynamics switches from the oscillatory to the excitable regime as the DIFICI waves disappear in the experiment.

  2. MicroRNAs in Amoebozoa: Deep sequencing of the small RNA population in the social amoeba Dictyostelium discoideum reveals developmentally regulated microRNAs

    PubMed Central

    Avesson, Lotta; Reimegård, Johan; Wagner, E. Gerhart H.; Söderbom, Fredrik

    2012-01-01

    The RNA interference machinery has served as a guardian of eukaryotic genomes since the divergence from prokaryotes. Although the basic components have a shared origin, silencing pathways directed by small RNAs have evolved in diverse directions in different eukaryotic lineages. Micro (mi)RNAs regulate protein-coding genes and play vital roles in plants and animals, but less is known about their functions in other organisms. Here, we report, for the first time, deep sequencing of small RNAs from the social amoeba Dictyostelium discoideum. RNA from growing single-cell amoebae as well as from two multicellular developmental stages was sequenced. Computational analyses combined with experimental data reveal the expression of miRNAs, several of them exhibiting distinct expression patterns during development. To our knowledge, this is the first report of miRNAs in the Amoebozoa supergroup. We also show that overexpressed miRNA precursors generate miRNAs and, in most cases, miRNA* sequences, whose biogenesis is dependent on the Dicer-like protein DrnB, further supporting the presence of miRNAs in D. discoideum. In addition, we find miRNAs processed from hairpin structures originating from an intron as well as from a class of repetitive elements. We believe that these repetitive elements are sources for newly evolved miRNAs. PMID:22875808

  3. Isolation of two novel ras genes in Dictyostelium discoideum; evidence for a complex, developmentally regulated ras gene subfamily.

    PubMed

    Daniel, J; Bush, J; Cardelli, J; Spiegelman, G B; Weeks, G

    1994-02-01

    In Dictyostelium discoideum, three ras genes (rasD, rasG and rasB) and one ras-related gene (rap1) have been previously isolated and characterized, and the deduced amino acid sequence of their predicted protein products share at least 50% sequence identity with the human H-Ras protein. We have now cloned and characterized two additional members of the ras gene subfamily in Dictyostelium, rasC and rasS. These genes are developmentally regulated and unlike the previously isolated Dictyostelium ras genes, maximum levels of their transcripts were detected during aggregation, suggesting that the encoded proteins have distinct functions during aggregation. The rasC cDNA encodes a 189 amino acid protein that is 65% identical to the Dictyostelium RasD and RasG proteins and 56% identical to the human H-Ras protein. The predicted 194 amino acid gene product encoded by rasS is 60% identical to the Dictyostelium RasD and RasG proteins and 54% identical to the human H-Ras protein. Whereas RasD, RasG, RasB and Rap1 are totally conserved in their putative effector domains relative to H-Ras, RasC and RasS have single amino acid substitutions in their effector domains, consistent with the idea that they have unique functions. In RasC, aspartic acid-38 has been replaced by asparagine (D38N), and in RasS, isoleucine-36 has been replaced by leucine (I36L). In addition, both proteins have several differences in the effector-proximal domain, a domain which is believed to play a role in Ras target activation. In RasC, there is a single conservative amino acid change in the canonical sequence of the binding site for the Ras-specific monoclonal antibody Y13-259, and consequently, RasC is less immunoreactive with the antibody than either of the Dictyostelium RasD or RasG proteins. In contrast, RasS, which has three substitutions in the Y13-259 binding site, does not react with the Y13-259 antibody.

  4. Relevance of the bioavailable fraction of DDT and its metabolites in freshwater sediment toxicity: New insight into the mode of action of these chemicals on Dictyostelium discoideum.

    PubMed

    Sforzini, Susanna; Governa, Daniela; Boeri, Marta; Oliveri, Laura; Oldani, Alessandro; Vago, Fabio; Viarengo, Aldo; Borrelli, Raffaella

    2016-10-01

    In this work, the toxicity of lake sediments contaminated with DDT and its metabolites DDD and DDE (collectively, DDX) was evaluated with widely used toxicity tests (i.e., Vibrio fischeri, Daphnia magna, Pseudokirchneriella subcapitata, and Lumbriculus variegatus) and with the social amoeba Dictyostelium discoideum, a model organism that is also suitable for studying pollutant-induced alterations at the molecular and cellular levels. Although the DDX concentration in the sediments was high (732.5 ppb), the results suggested a minimal environmental risk; in fact, no evidence of harmful effects was found using the different bioassays or when we considered the results of more sensitive sublethal biomarkers in D. discoideum amoebae. In line with the biological results, the chemical data showed that the concentration of DDX in the pore water (in general a highly bioavailable phase) showed a minimal value (0.0071ppb). To confirm the importance of the bioavailability of the toxic chemicals in determining their biological effects and to investigate the mechanisms of DDX toxicity, we exposed D. discoideum amoebae to 732.5ppb DDX in water solution. DDX had no effect on cell viability; however, a strong reduction in amoebae replication rate was observed, which depended mainly on a reduction in endocytosis rate and on lysosomal and mitochondrial alterations. In the presence of a moderate and transient increase in reactive oxygen species, the glutathione level in DDX-exposed amoebae drastically decreased. These results highlight that studies of the bioavailability of pollutants in environmental matrices and their biological effects are essential for site-specific ecological risk assessment. Moreover, glutathione depletion in DDX-exposed organisms is a new finding that could open the possibility of developing new pesticide mixtures that are more effective against DDT-resistant malaria vectors.

  5. Naringenin is a novel inhibitor of Dictyostelium cell proliferation and cell migration

    SciTech Connect

    Russ, Misty; Martinez, Raquel; Ali, Hind; Steimle, Paul A. . E-mail: p_steiml@uncg.edu

    2006-06-23

    Naringenin is a flavanone compound that alters critical cellular processes such as cell multiplication, glucose uptake, and mitochondrial activity. In this study, we used the social amoeba, Dictyostelium discoideum, as a model system for examining the cellular processes and signaling pathways affected by naringenin. We found that naringenin inhibited Dictyostelium cell division in a dose-dependent manner (IC{sub 5} {approx} 20 {mu}M). Assays of Dictyostelium chemotaxis and multicellular development revealed that naringenin possesses a previously unrecognized ability to suppress amoeboid cell motility. We also found that naringenin, which is known to inhibit phosphatidylinositol 3-kinase activity, had no apparent effect on phosphatidylinositol 3,4,5-trisphosphate synthesis in live Dictyostelium cells; suggesting that this compound suppresses cell growth and migration via alternative signaling pathways. In another context, the discoveries described here highlight the value of using the Dictyostelium model system for identifying and characterizing the mechanisms by which naringenin, and related compounds, exert their effects on eukaryotic cells.

  6. Fluorographic detection of tritiated glycopeptides and oligosaccharides separated on polyacrylamide gels: analysis of glycans from Dictyostelium discoideum glycoproteins

    SciTech Connect

    Prem Das, O.; Henderson, E.J.

    1986-11-01

    Previous workers have shown that oligosaccharides and glycopeptides can be separated by electrophoresis in buffers containing borate ions. However, normal fluorography of tritium-labeled structures cannot be performed because the glycans are soluble and can diffuse during equilibration with scintillants. This problem has been circumvented by equilibration of the gel with 2,5-diphenyloxazole (PPO) prior to electrophoresis. The presence of PPO in the gel during electrophoresis does not alter mobility of the glycopeptides and oligosaccharides. After electrophoresis, the gel is simply dried and fluorography performed. This allows sensitive and precise comparisons of labeled samples in parallel lanes of a slab gel and, since mobilities are highly reproducible, between different gels. The procedure is preparative in that after fluorography the gel bands can be quantitatively eluted for further study, without any apparent modification by the procedure. In this report, the procedure is illustrated by fractionation of both neutral and anionic glycopeptides produced by the cellular slime mold Dictyostelium discoideum.

  7. Structure of the N-terminal domain of the adenylyl cyclase-associated protein (CAP) from Dictyostelium discoideum.

    PubMed

    Ksiazek, Dorota; Brandstetter, Hans; Israel, Lars; Bourenkov, Gleb P; Katchalova, Galina; Janssen, Klaus-Peter; Bartunik, Hans D; Noegel, Angelika A; Schleicher, Michael; Holak, Tad A

    2003-09-01

    Cyclase-associated proteins (CAPs) are widely distributed and highly conserved proteins that regulate actin remodeling in response to cellular signals. The N termini of CAPs play a role in Ras signaling and bind adenylyl cyclase; the C termini bind to G-actin and thereby alter the dynamic rearrangements of the microfilament system. We report here the X-ray structure of the core of the N-terminal domain of the CAP from Dictyostelium discoideum, which comprises residues 51-226, determined by a combination of single isomorphous replacement with anomalous scattering (SIRAS). The overall structure of this fragment is an alpha helix bundle composed of six antiparallel helices. Results from gel filtration and crosslinking experiments for CAP(1-226), CAP(255-464), and the full-length protein, together with the CAP N-terminal domain structure and the recently determined CAP C-terminal domain structure, provide evidence that the functional structure of CAP is multimeric.

  8. NMR structural characterization of the N-terminal domain of the adenylyl cyclase-associated protein (CAP) from Dictyostelium discoideum.

    PubMed

    Mavoungou, Chrystelle; Israel, Lars; Rehm, Till; Ksiazek, Dorota; Krajewski, Marcin; Popowicz, Grzegorz; Noegel, Angelika A; Schleicher, Michael; Holak, Tad A

    2004-05-01

    Cyclase-associated proteins (CAPs) are highly conserved, ubiquitous actin binding proteins that are involved in microfilament reorganization. The N-termini of CAPs play a role in Ras signaling and bind adenylyl cyclase; the C-termini bind to G-actin. We report here the NMR characterization of the amino-terminal domain of CAP from Dictyostelium discoideum (CAP(1-226)). NMR data, including the steady state (1)H-(15)N heteronuclear NOE experiments, indicate that the first 50 N-terminal residues are unstructured and that this highly flexible serine-rich fragment is followed by a stable, folded core starting at Ser 51. The NMR structure of the folded core is an alpha-helix bundle composed of six antiparallel helices, in a stark contrast to the recently determined CAP C-terminal domain structure, which is solely built by beta-strands.

  9. Flow-Driven Waves and Phase-Locked Self-Organization in Quasi-One-Dimensional Colonies of Dictyostelium discoideum

    NASA Astrophysics Data System (ADS)

    Gholami, A.; Steinbock, O.; Zykov, V.; Bodenschatz, E.

    2015-01-01

    We report experiments on flow-driven waves in a microfluidic channel containing the signaling slime mold Dictyostelium discoideum. The observed cyclic adenosine monophosphate (cAMP) wave trains developed spontaneously in the presence of flow and propagated with the velocity proportional to the imposed flow velocity. The period of the wave trains was independent of the flow velocity. Perturbations of flow-driven waves via external periodic pulses of the signaling agent cAMP induced 1 ∶1 , 2 ∶1 , 3 ∶1 , and 1 ∶2 frequency responses, reminiscent of Arnold tongues in forced oscillatory systems. We expect our observations to be generic to active media governed by reaction-diffusion-advection dynamics, where spatially bound autocatalytic processes occur under flow conditions.

  10. Pseudopodium extension and amoeboid locomotion in Dictyostelium discoideum: Possible autowave behaviour of F-actin

    NASA Astrophysics Data System (ADS)

    Vicker, Michael G.; Xiang, Wei; Plath, Peter J.; Wosniok, Werner

    1997-02-01

    Supramolecular patterns of filamentous (F-)actin up to several micrometres across were visualized within projections of locomotory amoebae after cell fixation and staining with phalloidin-rhodamine. The patterns included rings, single and double spirals, some apparently colliding and disintegrating. Cell stimulation with a pulse of the chemoattractant cyclic AMP induced damping oscillations in F-actin ring frequency with a period of 6-7 s. Ring front propagation after stimulation was modelled by Markov and Fourier methods at 3.1-17.5 μm/min, similar to actual cell speed. We argue that the dynamics and detailed morphological correspondence of these F-actin structures to wave patterns in chemical reaction-diffusion systems strongly supports the interpretation that Dictyostelium cytoplasm behaves as an unstable, excitable medium enabling the propagation of self-organized, physico-chemical relaxation oscillations, i.e. autowaves, of reversible F-actin assembly or aggregation - a new state of actin - fundamental to pseudopodium extension, cell locomotion, chemotaxis and other cell functions.

  11. Amoeba-resisting bacteria found in multilamellar bodies secreted by Dictyostelium discoideum: social amoebae can also package bacteria.

    PubMed

    Paquet, Valérie E; Charette, Steve J

    2016-03-01

    Many bacteria can resist phagocytic digestion by various protozoa. Some of these bacteria (all human pathogens) are known to be packaged in multilamellar bodies produced in the phagocytic pathway of the protozoa and that are secreted into the extracellular milieu. Packaged bacteria are protected from harsh conditions, and the packaging process is suspected to promote bacterial persistence in the environment. To date, only a limited number of protozoa, belonging to free-living amoebae and ciliates, have been shown to perform bacteria packaging. It is still unknown if social amoebae can do bacteria packaging. The link between the capacity of 136 bacterial isolates to resist the grazing of the social amoeba Dictyostelium discoideum and to be packaged by this amoeba was investigated in the present study. The 45 bacterial isolates displaying a resisting phenotype were tested for their capacity to be packaged. A total of seven isolates from Cupriavidus, Micrococcus, Microbacterium and Rathayibacter genera seemed to be packaged and secreted by D. discoideum based on immunofluorescence results. Electron microscopy confirmed that the Cupriavidus and Rathayibacter isolates were formally packaged. These results show that social amoebae can package some bacteria from the environment revealing a new aspect of microbial ecology.

  12. The TOM Complex of Amoebozoans: the Cases of the Amoeba Acanthamoeba castellanii and the Slime Mold Dictyostelium discoideum.

    PubMed

    Wojtkowska, Małgorzata; Buczek, Dorota; Stobienia, Olgierd; Karachitos, Andonis; Antoniewicz, Monika; Slocinska, Małgorzata; Makałowski, Wojciech; Kmita, Hanna

    2015-07-01

    Protein import into mitochondria requires a wide variety of proteins, forming complexes in both mitochondrial membranes. The TOM complex (translocase of the outer membrane) is responsible for decoding of targeting signals, translocation of imported proteins across or into the outer membrane, and their subsequent sorting. Thus the TOM complex is regarded as the main gate into mitochondria for imported proteins. Available data indicate that mitochondria of representative organisms from across the major phylogenetic lineages of eukaryotes differ in subunit organization of the TOM complex. The subunit organization of the TOM complex in the Amoebozoa is still elusive, so we decided to investigate its organization in the soil amoeba Acanthamoeba castellanii and the slime mold Dictyostelium discoideum. They represent two major subclades of the Amoebozoa: the Lobosa and Conosa, respectively. Our results confirm the presence of Tom70, Tom40 and Tom7 in the A. castellanii and D. discoideum TOM complex, while the presence of Tom22 and Tom20 is less supported. Interestingly, the Tom proteins display the highest similarity to Opisthokonta cognate proteins, with the exception of Tom40. Thus representatives of two major subclades of the Amoebozoa appear to be similar in organization of the TOM complex, despite differences in their lifestyle.

  13. Identification of a signal transduction response sequence element necessary for induction of a Dictyostelium discoideum gene by extracellular cyclic AMP.

    PubMed Central

    Pavlovic, J; Haribabu, B; Dottin, R P

    1989-01-01

    The signal transduction pathways that lead to gene induction are being intensively investigated in Dictyostelium discoideum. We have identified by deletion and transformation analysis a sequence element necessary for induction of a gene coding for uridine diphosphoglucose pyrophosphorylase (UDPGP1) of D. discoideum in response to extracellular cyclic AMP (cAMP). This regulatory element is located 380 base pairs upstream of the transcription start site and contains a G+C-rich partially palindromic sequence. It is not required for transcription per se but is required for induction of the gene in response to the stimulus of extracellular cAMP. The cAMP response sequence is also required for induction of the gene during normal development. A second A+T-rich cis-acting region located immediately downstream of the cAMP response sequence appears to be essential for the basal level of expression of the UDPGP1 gene. The position of the cAMP response element coincides with a DNase I-hypersensitive site that is observed when the UDPGP1 gene is actively transcribed. Images PMID:2557538

  14. dictyExpress: a Dictyostelium discoideum gene expression database with an explorative data analysis web-based interface

    PubMed Central

    Rot, Gregor; Parikh, Anup; Curk, Tomaz; Kuspa, Adam; Shaulsky, Gad; Zupan, Blaz

    2009-01-01

    Background Bioinformatics often leverages on recent advancements in computer science to support biologists in their scientific discovery process. Such efforts include the development of easy-to-use web interfaces to biomedical databases. Recent advancements in interactive web technologies require us to rethink the standard submit-and-wait paradigm, and craft bioinformatics web applications that share analytical and interactive power with their desktop relatives, while retaining simplicity and availability. Results We have developed dictyExpress, a web application that features a graphical, highly interactive explorative interface to our database that consists of more than 1000 Dictyostelium discoideum gene expression experiments. In dictyExpress, the user can select experiments and genes, perform gene clustering, view gene expression profiles across time, view gene co-expression networks, perform analyses of Gene Ontology term enrichment, and simultaneously display expression profiles for a selected gene in various experiments. Most importantly, these tasks are achieved through web applications whose components are seamlessly interlinked and immediately respond to events triggered by the user, thus providing a powerful explorative data analysis environment. Conclusion dictyExpress is a precursor for a new generation of web-based bioinformatics applications with simple but powerful interactive interfaces that resemble that of the modern desktop. While dictyExpress serves mainly the Dictyostelium research community, it is relatively easy to adapt it to other datasets. We propose that the design ideas behind dictyExpress will influence the development of similar applications for other model organisms. PMID:19706156

  15. Overproduction of the regulatory subunit of the cAMP-dependent protein kinase blocks the differentiation of Dictyostelium discoideum.

    PubMed Central

    Simon, M N; Driscoll, D; Mutzel, R; Part, D; Williams, J; Véron, M

    1989-01-01

    During the aggregation of Dictyostelium discoideum extracellular cAMP is known to act as a chemotractant and as an inducer of cellular differentiation. However, its intracellular role as a second messenger remains obscure. We have constructed a fusion gene consisting of the cDNA encoding the regulatory subunit (R) of the cAMP-dependent protein kinase fused to the promoter and N-terminal-proximal sequences of a Dictyostelium actin gene. Stable transformants, containing multiple copies of this gene, overproduce the R subunit which accumulates prematurely relative to the endogenous protein. These transformants fail to aggregate. Detailed analysis has shown that they are blocked at interphase, the period prior to aggregation, and that they are severely defective in most responses to cAMP including the induction of gene expression. Our observations suggest that intracellular cAMP acts, presumably by activation of the catalytic subunit of the cAMP-dependent protein kinase, to facilitate early development. Images PMID:2551673

  16. Clues to γ-secretase, huntingtin and Hirano body normal function using the model organism Dictyostelium discoideum.

    PubMed

    Myre, Michael A

    2012-04-10

    Many neurodegenerative disorders, although related by their destruction of brain function, display remarkable cellular and/or regional pathogenic specificity likely due to a deregulated functionality of the mutant protein. However, neurodegenerative disease genes, for example huntingtin (HTT), the ataxins, the presenilins (PSEN1/PSEN2) are not simply localized to neurons but are ubiquitously expressed throughout peripheral tissues; it is therefore paramount to properly understand the earliest precipitating events leading to neuronal pathogenesis to develop effective long-term therapies. This means, in no unequivocal terms, it is crucial to understand the gene's normal function. Unfortunately, many genes are often essential for embryogenesis which precludes their study in whole organisms. This is true for HTT, the β-amyloid precursor protein (APP) and presenilins, responsible for early onset Alzheimer's disease (AD). To better understand neurological disease in humans, many lower and higher eukaryotic models have been established. So the question arises: how reasonable is the use of organisms to study neurological disorders when the model of choice does not contain neurons? Here we will review the surprising, and novel emerging use of the model organism Dictyostelium discoideum, a species of soil-living amoeba, as a valuable biomedical tool to study the normal function of neurodegenerative genes. Historically, the evidence on the usefulness of simple organisms to understand the etiology of cellular pathology cannot be denied. But using an organism without a central nervous system to understand diseases of the brain? We will first introduce the life cycle of Dictyostelium, the presence of many disease genes in the genome and how it has provided unique opportunities to identify mechanisms of disease involving actin pathologies, mitochondrial disease, human lysosomal and trafficking disorders and host-pathogen interactions. Secondly, I will highlight recent studies on

  17. Clues to γ-secretase, huntingtin and Hirano body normal function using the model organism Dictyostelium discoideum

    PubMed Central

    2012-01-01

    Many neurodegenerative disorders, although related by their destruction of brain function, display remarkable cellular and/or regional pathogenic specificity likely due to a deregulated functionality of the mutant protein. However, neurodegenerative disease genes, for example huntingtin (HTT), the ataxins, the presenilins (PSEN1/PSEN2) are not simply localized to neurons but are ubiquitously expressed throughout peripheral tissues; it is therefore paramount to properly understand the earliest precipitating events leading to neuronal pathogenesis to develop effective long-term therapies. This means, in no unequivocal terms, it is crucial to understand the gene's normal function. Unfortunately, many genes are often essential for embryogenesis which precludes their study in whole organisms. This is true for HTT, the β-amyloid precursor protein (APP) and presenilins, responsible for early onset Alzheimer's disease (AD). To better understand neurological disease in humans, many lower and higher eukaryotic models have been established. So the question arises: how reasonable is the use of organisms to study neurological disorders when the model of choice does not contain neurons? Here we will review the surprising, and novel emerging use of the model organism Dictyostelium discoideum, a species of soil-living amoeba, as a valuable biomedical tool to study the normal function of neurodegenerative genes. Historically, the evidence on the usefulness of simple organisms to understand the etiology of cellular pathology cannot be denied. But using an organism without a central nervous system to understand diseases of the brain? We will first introduce the life cycle of Dictyostelium, the presence of many disease genes in the genome and how it has provided unique opportunities to identify mechanisms of disease involving actin pathologies, mitochondrial disease, human lysosomal and trafficking disorders and host-pathogen interactions. Secondly, I will highlight recent studies on

  18. Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

    PubMed Central

    Malinovska, Liliana; Alberti, Simon

    2016-01-01

    The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we showed that D. discoideum is remarkably resilient to protein aggregation and can be used to gain insights into the cellular protein quality control system. However, the use of D. discoideum as a model system poses several challenges to microscopy-based experimental approaches, such as the high motility of the cells and their susceptibility to photo-toxicity. The latter proves to be especially challenging when studying protein homeostasis, as the phototoxic effects can induce a cellular stress response and thus alter to behavior of the protein quality control system. Temperature increase is a commonly used way to induce cellular stress. Here, we describe a temperature-controllable imaging protocol, which allows observing temperature-induced perturbations in D. discoideum. Moreover, when applied at normal growth temperature, this imaging protocol can also noticeably reduce photo-toxicity, thus allowing imaging with higher intensities. This can be particularly useful when imaging proteins with very low expression levels. Moreover, the high mobility of the cells often requires the acquisition of multiple fields of view to follow individual cells, and the number of fields needs to be balanced against the desired time interval and exposure time. PMID:28060267

  19. 1H, 15N and 13C assignments of domain 5 of Dictyostelium discoideum gelation factor (ABP-120) in its native and 8M urea-denatured states.

    PubMed

    Hsu, Shang-Te Danny; Cabrita, Lisa D; Christodoulou, John; Dobson, Christopher M

    2009-06-01

    The gelation factor from Dictyostelium discoideum (ABP-120) is an actin binding protein consisting of six immunoglobulin (Ig) domains in the C-terminal rod domain. We have recently used the pair of domains 5 and 6 of ABP-120 as a model system for studying multi-domain nascent chain folding on the ribosome. Here we present the NMR assignments of domain 5 in its native and 8M urea-denatured states.

  20. Salmonella typhimurium is pathogenic for Dictyostelium cells and subverts the starvation response.

    PubMed

    Sillo, Alessio; Matthias, Jan; Konertz, Roman; Bozzaro, Salvatore; Eichinger, Ludwig

    2011-11-01

    In unicellular amoebae, such as Dictyostelium discoideum, bacterial phagocytosis is a food hunting device, while in higher organisms it is the first defence barrier against microbial infection. In both cases, pathogenic bacteria exploit phagocytosis to enter the cell and multiply intracellularly. Salmonella typhimurium, the agent of food-borne gastroenteritis, is phagocytosed by both macrophages and Dictyostelium cells. By using cell biological assays and global transcriptional analysis with DNA microarrays covering the Dictyostelium genome, we show here that S. typhimurium is pathogenic for Dictyostelium cells. Depending on the degree of virulence, which in turn depended on bacterial growth conditions, Salmonella could kill Dictyostelium cells or inhibit their growth and development. In the early phase of infection in non-nutrient buffer, the ingested bacteria escaped degradation, induced a starvation-like transcriptional response but inhibited selectively genes required for chemotaxis and aggregation. This way differentiation of the host cells into spore and stalk cells was blocked or delayed, which in turn is likely to be favourable for the establishment of a replicative niche for Salmonella. Inhibition of the aggregation competence and chemotactic streaming of aggregation-competent cells in the presence of Salmonella suggests interference with cAMP signalling.

  1. Two ras genes in Dictyostelium minutum show high sequence homology, but different developmental regulation from Dictyostelium discoideum rasD and rasG genes.

    PubMed

    van Es, S; Kooistra, R A; Schaap, P

    1997-03-10

    The social amoeba Dictyostelium discoideum expresses five ras genes at different stages of development. One of them, DdrasD is expressed during postaggregative development and transcription is induced by extracellular cAMP. A homologue of DdrasD, the DdrasG gene, is expressed exclusively during vegetative growth. We cloned two ras homologues Dmras1 and Dmras2 from the primitive species D. minutum, which show high homology to DdrasD and DdrasG and less homology to the other Ddras genes. In contrast to the DdrasD and DdrasG genes, both the Dmras1 and Dmras2 genes are expressed during the entire course of development. The expression levels are low during growth, increase at the onset of starvation and do not decrease until fruiting bodies have formed. Expression of neither Dmras1 or Dmras2 is regulated by cAMP. So even though the high degree of homology between the ras genes of different species suggests conservation of function, this function is apparently not associated with a specific developmental stage.

  2. The Effects of Extracellular Calcium on Motility, Pseudopod and Uropod Formation, Chemotaxis and the Cortical Localization of Myosin II in Dictyostelium discoideum

    PubMed Central

    Lusche, Daniel F.; Wessels, Deborah; Soll, David R.

    2009-01-01

    Extracellular Ca++, a ubiquitous cation in the soluble environment of cells both free living and within the human body, regulates most aspects of amoeboid cell motility, including shape, uropod formation, pseudopod formation, velocity and turning in Dictyostelium discoideum. Hence it affects the efficiency of both basic motile behavior and chemotaxis. Extracellular Ca++ is optimal at 10 mM. A gradient of the chemoattractant cAMP generated in the absence of added Ca++ only affects turning, but in combination with extracellular Ca++, enhances the effects of extracellular Ca++. Potassium, at 40 mM, can substitute for Ca++. Mg++, Mn++, Zn++ and Na+ cannot. Extracellular Ca++, or K+, also induce the cortical localization of myosin II in a polar fashion. The effects of Ca++, K+ or a cAMP gradient do not appear to be similarly mediated by an increase in the general pool of free cytosolic Ca++. These results suggest a model, in which each agent functioning through different signaling systems, converge to affect the cortical localization of myosin II, which in turn effects the behavioral changes leading to efficient cell motility and chemotaxis. PMID:19363786

  3. Simple system--substantial share: the use of Dictyostelium in cell biology and molecular medicine.

    PubMed

    Müller-Taubenberger, Annette; Kortholt, Arjan; Eichinger, Ludwig

    2013-02-01

    Dictyostelium discoideum offers unique advantages for studying fundamental cellular processes, host-pathogen interactions as well as the molecular causes of human diseases. The organism can be easily grown in large amounts and is amenable to diverse biochemical, cell biological and genetic approaches. Throughout their life cycle Dictyostelium cells are motile, and thus are perfectly suited to study random and directed cell motility with the underlying changes in signal transduction and the actin cytoskeleton. Dictyostelium is also increasingly used for the investigation of human disease genes and the crosstalk between host and pathogen. As a professional phagocyte it can be infected with several human bacterial pathogens and used to study the infection process. The availability of a large number of knock-out mutants renders Dictyostelium particularly useful for the elucidation and investigation of host cell factors. A powerful armory of molecular genetic techniques that have been continuously expanded over the years and a well curated genome sequence, which is accessible via the online database dictyBase, considerably strengthened Dictyostelium's experimental attractiveness and its value as model organism.

  4. The NMRA/NMRAL1 homologue PadA modulates the expression of extracellular cAMP relay genes during aggregation in Dictyostelium discoideum.

    PubMed

    Garciandia, Ane; Suarez, Teresa

    2013-09-15

    NMRA-like proteins belong to a class of conserved transcriptional regulators that function as direct sensors of the metabolic state of the cell and link basic metabolism to changes in gene expression. PadA was the first NMRA-like protein described in Dictyostelium discoideum and was shown to be necessary for prestalk cell differentiation and correct development. We describe and characterize padA(-) mutant phenotype during the onset of development, which results in the formation of abnormally small territories and impairment of cAMP responses. Transcriptional analysis shows that cAMP-induced gene expression is downregulated in padA(-), particularly the genes that establish the extracellular cAMP relay. The mutant phenotype can be rescued with the constitutive expression of one of these genes, carA, encoding the cAMP receptor. Transcriptional analysis of padA(-)/A15::carA showed that carA maximum mRNA levels were not reached during aggregation. Our data support a regulatory role for PadA on the regulation of extracellular cAMP relay genes during aggregation and suggest that PadA is required to achieve carA full induction.

  5. Systematic analysis of γ-aminobutyric acid (GABA) metabolism and function in the social amoeba Dictyostelium discoideum.

    PubMed

    Wu, Yuantai; Janetopoulos, Chris

    2013-05-24

    While GABA has been suggested to regulate spore encapsulation in the social amoeba Dictyostelium discoideum, the metabolic profile and other potential functions of GABA during development remain unclear. In this study, we investigated the homeostasis of GABA metabolism by disrupting genes related to GABA metabolism and signaling. Extracellular levels of GABA are tightly regulated during early development, and GABA is generated by the glutamate decarboxylase, GadB, during growth and in early development. However, overexpression of the prespore-specific homologue, GadA, in the presence of GadB reduces production of extracellular GABA. Perturbation of extracellular GABA levels delays the process of aggregation. Cytosolic GABA is degraded by the GABA transaminase, GabT, in the mitochondria. Disruption of a putative vesicular GABA transporter (vGAT) homologue DdvGAT reduces secreted GABA. We identified the GABAB receptor-like family member GrlB as the major GABA receptor during early development, and either disruption or overexpression of GrlB delays aggregation. This delay is likely the result of an abolished pre-starvation response and late expression of several "early" developmental genes. Distinct genes are employed for GABA generation during sporulation. During sporulation, GadA alone is required for generating GABA and DdvGAT is likely responsible for GABA secretion. GrlE but not GrlB is the GABA receptor during late development.

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

    PubMed

    Shimada, Nao; Kawata, Takefumi

    2007-06-01

    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. There were 12 multicopy suppressors of Dd-STATa, of which 4 encoded segments of a known noncoding RNA, dutA. Expression of dutA is specific to the pstA zone, the region where Dd-STATa is activated. In suppressed strains the expression patterns of several putative Dd-STATa target genes become similar to the wild-type strain. In addition, the amount of the tyrosine-phosphorylated form of Dd-STATa is significantly increased in the suppressed strain. These results indicate that partial copies of dutA may act upstream of Dd-STATa to regulate tyrosine phosphorylation by an unknown mechanism.

  7. Expression and organization of BP74, a cyclic AMP-regulated gene expressed during Dictyostelium discoideum development.

    PubMed Central

    Hopkinson, S B; Pollenz, R S; Drummond, I; Chisholm, R L

    1989-01-01

    We have characterized a cDNA and the corresponding gene for a cyclic AMP-inducible gene expressed during Dictyostelium development. This gene, BP74, was found to be first expressed about the time of aggregate formation, approximately 6 h after starvation. Accumulation of BP74 mRNA did not occur in Dictyostelium cells that had been starved in fast-shaken suspension cultures but was induced in similar cultures to which cyclic AMP pulses had been added. The BP74 cDNA and gene were characterized by DNA sequence analysis and transcriptional mapping. When the BP74 promoter region was fused with a chloramphenicol acetyltransferase reporter gene and reintroduced into Dictyostelium cells, the transfected chloramphenicol acetyltransferase gene displayed the same developmentally regulated pattern of expression as did the endogenous BP74 gene, suggesting that all of the cis-acting elements required for regulated expression were carried by a 2-kilobase cloned genomic fragment. On the basis of sequence analysis, the gene appeared to encode a protein containing a 20-residue hydrophobic sequence at the amino-terminal end and 26 copies of a 20-amino-acid repeat. Images PMID:2555685

  8. Properties of a non-bioactive fluorescent derivative of differentiation-inducing factor-3, an anti-tumor agent found in Dictyostelium discoideum

    PubMed Central

    Kubohara, Yuzuru; Kikuchi, Haruhisa; Matsuo, Yusuke; Oshima, Yoshiteru; Homma, Yoshimi

    2014-01-01

    ABSTRACT Differentiation-inducing factor-3 (DIF-3), found in the cellular slime mold Dictyostelium discoideum, and its derivatives, such as butoxy-DIF-3 (Bu-DIF-3), are potent anti-tumor agents. To investigate the activity of DIF-like molecules in tumor cells, we recently synthesized a green fluorescent DIF-3 derivative, BODIPY-DIF-3G, and analyzed its bioactivity and cellular localization. In this study, we synthesized a red (orange) fluorescent DIF-3 derivative, BODIPY-DIF-3R, and compared the cellular localization and bioactivities of the two BODIPY-DIF-3s in HeLa human cervical cancer cells. Both fluorescent compounds penetrated the extracellular membrane within 0.5 h and localized mainly to the mitochondria. In formalin-fixed cells, the two BODIPY-DIF-3s also localized to the mitochondria, indicating that the BODIPY-DIF-3s were incorporated into mitochondria independently of the mitochondrial membrane potential. After treatment for 3 days, BODIPY-DIF-3G, but not BODIPY-DIF-3R, induced mitochondrial swelling and suppressed cell proliferation. Interestingly, the swollen mitochondria were stainable with BODIPY-DIF-3G but not with BODIPY-DIF-3R. When added to isolated mitochondria in vitro, BODIPY-DIF-3G increased dose-dependently the rate of O2 consumption, but BODIPY-DIF-3R did not. These results suggest that the bioactive BODIPY-DIF-3G suppresses cell proliferation, at least in part, by altering mitochondrial activity, whereas the non-bioactive BODIPY-DIF-3R localizes to the mitochondria but does not affect mitochondrial activity or cell proliferation. PMID:24682009

  9. The Effects of Temperature Variation on the Sensitivity to Pesticides: a Study on the Slime Mould Dictyostelium discoideum (Protozoa).

    PubMed

    Amaroli, Andrea

    2015-07-01

    Slime moulds live in agricultural ecosystems, where they play an important role in the soil fertilization and in the battle against crop pathogens. In an agricultural soil, the amoebae are exposed to different stress factors such as pesticides and weather conditions. The use of pesticides increased up from 0.49 kg per hectare in 1961 to 2 kg in 2004, and the global greenhouse gas emission has grown 70% between 1970 and 2004 leading to a global fluctuation of average surface temperature. Therefore, the European Directive 2009/128/EC has led to a new approach to agriculture, with the transition from an old concept based on high use of pesticides and fossil fuels to an agriculture aware of biodiversity and health issues. We studied the effects of temperature variations and pesticides on Dictyostelium discoideum. We measured the fission rate, the ability to differentiate and the markers of stress such as the activity and presence of pseudocholinesterase and the presence of heat shock protein 70. Our results highlight how the sensitivity to zinc, aluminium, silver, copper, cadmium, mercury, diazinon and dicofol changes for a 2 °C variation from nothing/low to critical. Our work suggests considering, in future regulations, about the use of pesticides as their toxic effect on non-target organisms is strongly influenced by climate temperatures. In addition, there is a need for a new consideration of the protozoa, which takes into account recent researches about the presence in this microorganism of classical neurotransmitters that, similar to those in animals, make protozoa an innocent target of neurotoxic pesticides in the battle against the pest crops.

  10. Structure, dynamics and folding of an immunoglobulin domain of the gelation factor (ABP-120) from Dictyostelium discoideum.

    PubMed

    Hsu, Shang-Te Danny; Cabrita, Lisa D; Fucini, Paola; Dobson, Christopher M; Christodoulou, John

    2009-05-15

    We have carried out a detailed structural and dynamical characterisation of the isolated fifth repeat of the gelation factor (ABP-120) from Dictyostelium discoideum (ddFLN5) by NMR spectroscopy to provide a basis for studies of co-translational folding on the ribosome of this immunoglobulin-like domain. The isolated ddFLN5 can fold autonomously in solution into a structure that resembles very closely the crystal structure of the domain in a construct in which the adjacent sixth repeat (ddFLN6) is covalently linked to its C-terminus in tandem but deviates locally from a second crystal structure in which ddFLN5 is flanked by ddFLN4 and ddFLN6 at both N- and C-termini. Conformational fluctuations were observed via (15)N relaxation methods and are primarily localised in the interstrand loops that encompass the C-terminal hemisphere. These fluctuations are distinct in location from the region where line broadening is observed in ddFLN5 when attached to the ribosome as part of a nascent chain. This observation supports the conclusion that the broadening is associated with interactions with the ribosome surface [Hsu, S. T. D., Fucini, P., Cabrita, L. D., Launay, H., Dobson, C. M. & Christodoulou, J. (2007). Structure and dynamics of a ribosome-bound nascent chain by NMR spectroscopy. Proc. Natl. Acad. Sci. USA, 104, 16516-16521]. The unfolding of ddFLN5 induced by high concentrations of urea shows a low population of a folding intermediate, as inferred from an intensity-based analysis, a finding that differs from that of ddFLN5 as a ribosome-bound nascent chain. These results suggest that interesting differences in detail may exist between the structure of the domain in isolation and when linked to the ribosome and between protein folding in vitro and the folding of a nascent chain as it emerges from the ribosome.

  11. A micromechanic study of cell polarity and plasma membrane cell body coupling in Dictyostelium.

    PubMed Central

    Merkel, R; Simson, R; Simson, D A; Hohenadl, M; Boulbitch, A; Wallraff, E; Sackmann, E

    2000-01-01

    We used micropipettes to aspirate leading and trailing edges of wild-type and mutant cells of Dictyostelium discoideum. Mutants were lacking either myosin II or talin, or both proteins simultaneously. Talin is a plasma membrane-associated protein important for the coupling between membrane and actin cortex, whereas myosin II is a cytoplasmic motor protein essential for the locomotion of Dictyostelium cells. Aspiration into the pipette occurred above a threshold pressure only. For all cells containing talin this threshold was significantly lower at the leading edge of an advancing cell as compared to its rear end, whereas we found no such difference in cells lacking talin. Wild-type and talin-deficient cells were able to retract from the pipette against an applied suction pressure. In these cells, retraction was preceded by an accumulation of myosin II in the tip of the aspirated cell lobe. Mutants lacking myosin II could not retract, even if the suction pressures were removed after aspiration. We interpreted the initial instability and the subsequent plastic deformation of the cell surface during aspiration in terms of a fracture between the cell plasma membrane and the cell body, which may involve destruction of part of the cortex. Models are presented that characterize the coupling strength between membrane and cell body by a surface energy sigma. We find sigma approximately 0.6(1.6) mJ/m(2) at the leading (trailing) edge of wild-type cells. PMID:10920005

  12. Cell-to-cell coordination for the spontaneous cAMP oscillation in Dictyostelium

    NASA Astrophysics Data System (ADS)

    Nagano, Seido; Sakurai, Shunsuke

    2013-12-01

    We propose a new cellular dynamics scheme for the spontaneous cAMP oscillations in Dictyostelium discoideum. Our scheme seamlessly integrates both receptor dynamics and G-protein dynamics into our previously developed cellular dynamics scheme. Extensive computer simulation studies based on our new cellular dynamics scheme were conducted in mutant cells to evaluate the molecular network. The validity of our proposed molecular network as well as the controversial PKA-dependent negative feedback mechanism was supported by our simulation studies. Spontaneous cAMP oscillations were not observed in a single mutant cell. However, multicellular states of various mutant cells consistently initiated spontaneous cAMP oscillations. Therefore, cell-to-cell coordination via the cAMP receptor is essential for the robust initiation of spontaneous cAMP oscillations.

  13. A cell number-counting factor regulates the cytoskeleton and cell motility in Dictyostelium.

    PubMed

    Tang, Lei; Gao, Tong; McCollum, Catherine; Jang, Wonhee; Vicker, Michael G; Ammann, Robin R; Gomer, Richard H

    2002-02-05

    Little is known about how a morphogenetic rearrangement of a tissue is affected by individual cells. Starving Dictyostelium discoideum cells aggregate to form dendritic streams, which then break up into groups of approximately 2 x 10(4) cells. Cell number is sensed at this developmental stage by using counting factor (CF), a secreted complex of polypeptides. A high extracellular concentration of CF indicates that there is a large number of cells, which then causes the aggregation stream to break up. Computer simulations indicated that stream breakup could be caused by CF decreasing cell-cell adhesion and/or increasing cell motility, and we observed that CF does indeed decrease cell-cell adhesion. We find here that CF increases cell motility. In Dictyostelium, motility is mediated by actin and myosin. CF increases the amounts of polymerized actin and the ABP-120 actin-crosslinking protein. Partially inhibiting motility by using drugs that interfere with actin polymerization reduces stream dissipation, resulting in fewer stream breaks and thus larger groups. CF also potentiates the phosphorylation and redistribution of myosin while repressing its basal level of assembly. The computer simulations indicated that a narrower distribution of group sizes results when a secreted factor modulates both adhesion and motility. CF thus seems to induce the morphogenesis of streams into evenly sized groups by increasing actin polymerization, ABP-120 levels, and myosin phosphorylation and decreasing adhesion and myosin polymerization.

  14. A developmentally regulated membrane protein gene in Dictyostelium discoideum is also induced by heat shock and cold shock.

    PubMed Central

    Maniak, M; Nellen, W

    1988-01-01

    We have analyzed the expression of the Dictyostelium gene P8A7 which had been isolated as a cDNA clone from an early developmentally regulated gene. The single genomic copy generated two mRNAs which were subject to different control mechanisms: while one mRNA (P8A7S) was regulated like the cell-type-nonspecific late genes, the other one (P8A7L) was induced during development, when cells were allowed to attach to a substrate, and when cells were subjected to stress, such as heat shock and cadmium. Interestingly the same induction was also observed with cold shock. RNA processing was inhibited by heat and cold shock, leading to nuclear accumulation of a precursor. The translated region of the cDNA was common to both mRNAs and encoded an unusually hydrophobic peptide with the characteristics of a membrane protein. Images PMID:3336356

  15. A cell number-counting factor regulates the cytoskeleton and cell motility in Dictyostelium

    PubMed Central

    Tang, Lei; Gao, Tong; McCollum, Catherine; Jang, Wonhee; Vicker, Michael G.; Ammann, Robin R.; Gomer, Richard H.

    2002-01-01

    Little is known about how a morphogenetic rearrangement of a tissue is affected by individual cells. Starving Dictyostelium discoideum cells aggregate to form dendritic streams, which then break up into groups of ≈2 × 104 cells. Cell number is sensed at this developmental stage by using counting factor (CF), a secreted complex of polypeptides. A high extracellular concentration of CF indicates that there is a large number of cells, which then causes the aggregation stream to break up. Computer simulations indicated that stream breakup could be caused by CF decreasing cell–cell adhesion and/or increasing cell motility, and we observed that CF does indeed decrease cell–cell adhesion. We find here that CF increases cell motility. In Dictyostelium, motility is mediated by actin and myosin. CF increases the amounts of polymerized actin and the ABP-120 actin-crosslinking protein. Partially inhibiting motility by using drugs that interfere with actin polymerization reduces stream dissipation, resulting in fewer stream breaks and thus larger groups. CF also potentiates the phosphorylation and redistribution of myosin while repressing its basal level of assembly. The computer simulations indicated that a narrower distribution of group sizes results when a secreted factor modulates both adhesion and motility. CF thus seems to induce the morphogenesis of streams into evenly sized groups by increasing actin polymerization, ABP-120 levels, and myosin phosphorylation and decreasing adhesion and myosin polymerization. PMID:11818526

  16. Modeling actin waves in dictyostelium cells

    NASA Astrophysics Data System (ADS)

    Wasnik, Vaibhav; Mukhopadhyay, Ranjan

    2011-03-01

    Actin networks in living cells demonstrate a high capacity for self-organization and are responsible for the formation of a variety of structures such as lamellopodia, phagocytic cups, and cleavage furrows. Recent experiments have studied actin waves formed on the surface of dictyostelium cells that have been treated with a depolymerizing agent. These waves are believed to be physiologically important, for example, for the formation of phagocytic cups. We propose and study a minimal model, based on the dendritic nucleation of actin polymers, to explain the formation of these waves. This model can be extended to study the dynamics of the coupled actin-membrane system.

  17. The prokaryote messenger c-di-GMP triggers stalk cell differentiation in Dictyostelium.

    PubMed

    Chen, Zhi-hui; Schaap, Pauline

    2012-08-30

    Cyclic di-(3′:5′)-guanosine monophosphate (c-di-GMP) is a major prokaryote signalling intermediate that is synthesized by diguanylate cyclases and triggers sessility and biofilm formation. We detected the first eukaryote diguanylate cyclases in all major groups of Dictyostelia. On food depletion, Dictyostelium discoideum amoebas collect into aggregates, which first transform into migrating slugs and then into sessile fruiting structures. These structures consist of a spherical spore mass that is supported by a column of stalk cells and a basal disk. A polyketide, DIF-1, which induces stalk-like cells in vitro, was isolated earlier. However, its role in vivo proved recently to be restricted to basal disk formation. Here we show that the Dictyostelium diguanylate cyclase, DgcA, produces c-di-GMP as the morphogen responsible for stalk cell differentiation. Dictyostelium discoideum DgcA synthesized c-di-GMP in a GTP-dependent manner and was expressed at the slug tip, which is the site of stalk cell differentiation. Disruption of the DgcA gene blocked the transition from slug migration to fructification and the expression of stalk genes. Fructification and stalk formation were restored by exposing DgcA-null slugs to wild-type secretion products or to c-di-GMP. Moreover, c-di-GMP, but not cyclic di-(3′:5′)-adenosine monophosphate, induced stalk gene expression in dilute cell monolayers. Apart from identifying the long-elusive stalk-inducing morphogen, our work also identifies a role for c-di-GMP in eukaryotes.

  18. Copine A is expressed in prestalk cells and regulates slug phototaxis and thermotaxis in developing Dictyostelium

    PubMed Central

    Flegel, Kerry A.; Pineda, Jaimie M.; Smith, Tasha S.; Laszczyk, Ann M.; Price, Janet M.; Karasiewicz, Kristen M.; Damer, Cynthia K.

    2011-01-01

    Copines are calcium-dependent membrane-binding proteins found in many eukaryotic organisms. We are studying the function of copines using the model organism, Dictyostelium discoideum. When under starvation conditions, Dictyostelium cells aggregate into mounds that become migrating slugs, which can move toward light and heat before culminating into a fruiting body. Previously, we showed that Dictyostelium cells lacking the copine A (cpnA) gene are not able to form fruiting bodies and instead arrest at the slug stage. In this study, we compared the slug behavior of cells lacking the cpnA gene to the slug behavior of wild-type cells. The slugs formed by cpnA- cells were much larger than wild-type slugs and exhibited no phototaxis and negative thermotaxis in the same conditions that wild-type slugs exhibited positive phototaxis and thermotaxis. Mixing as little as 5% wild-type cells with cpnA- cells rescued the phototaxis and thermotaxis defects, suggesting that CpnA plays a specific role in the regulation of the production and/or release of a signaling molecule. Reducing extracellular levels of ammonia also partially rescued the phototaxis and thermotaxis defects of cpnA- slugs, suggesting that CpnA may have a specific role in regulating ammonia signaling. Expressing the lacZ gene under the cpnA promoter in wild-type cells indicated cpnA is preferentially expressed in the prestalk cells found in the anterior part of the slug, which include the cells at the tip of the slug that regulate phototaxis, thermotaxis, and the initiation of culmination into fruiting bodies. Our results suggest that CpnA plays a role in the regulation of the signaling pathways, including ammonia signaling, necessary for sensing and/or orienting toward light and heat in the prestalk cells of the Dictyostelium slug. PMID:21950343

  19. Copine A is expressed in prestalk cells and regulates slug phototaxis and thermotaxis in developing Dictyostelium.

    PubMed

    Flegel, Kerry A; Pineda, Jaimie M; Smith, Tasha S; Laszczyk, Ann M; Price, Janet M; Karasiewicz, Kristen M; Damer, Cynthia K

    2011-10-01

    Copines are calcium-dependent membrane-binding proteins found in many eukaryotic organisms. We are studying the function of copines using the model organism, Dictyostelium discoideum. When under starvation conditions, Dictyostelium cells aggregate into mounds that become migrating slugs, which can move toward light and heat before culminating into a fruiting body. Previously, we showed that Dictyostelium cells lacking the copine A (cpnA) gene are not able to form fruiting bodies and instead arrest at the slug stage. In this study, we compared the slug behavior of cells lacking the cpnA gene to the slug behavior of wild-type cells. The slugs formed by cpnA- cells were much larger than wild-type slugs and exhibited no phototaxis and negative thermotaxis in the same conditions that wild-type slugs exhibited positive phototaxis and thermotaxis. Mixing as little as 5% wild-type cells with cpnA- cells rescued the phototaxis and thermotaxis defects, suggesting that CpnA plays a specific role in the regulation of the production and/or release of a signaling molecule. Reducing extracellular levels of ammonia also partially rescued the phototaxis and thermotaxis defects of cpnA- slugs, suggesting that CpnA may have a specific role in regulating ammonia signaling. Expressing the lacZ gene under the cpnA promoter in wild-type cells indicated cpnA is preferentially expressed in the prestalk cells found in the anterior part of the slug, which include the cells at the tip of the slug that regulate phototaxis, thermotaxis, and the initiation of culmination into fruiting bodies. Our results suggest that CpnA plays a role in the regulation of the signaling pathways, including ammonia signaling, necessary for sensing and/or orienting toward light and heat in the prestalk cells of the Dictyostelium slug.

  20. A Model for Direction Sensing in Dictyostelium discoideum: Ras Activity and Symmetry Breaking Driven by a Gβγ-Mediated, Gα2-Ric8 -- Dependent Signal Transduction Network

    PubMed Central

    Cheng, Yougan; Othmer, Hans

    2016-01-01

    Chemotaxis is a dynamic cellular process, comprised of direction sensing, polarization and locomotion, that leads to the directed movement of eukaryotic cells along extracellular gradients. As a primary step in the response of an individual cell to a spatial stimulus, direction sensing has attracted numerous theoretical treatments aimed at explaining experimental observations in a variety of cell types. Here we propose a new model of direction sensing based on experiments using Dictyostelium discoideum (Dicty). The model is built around a reaction-diffusion-translocation system that involves three main component processes: a signal detection step based on G-protein-coupled receptors (GPCR) for cyclic AMP (cAMP), a transduction step based on a heterotrimetic G protein Gα2βγ, and an activation step of a monomeric G-protein Ras. The model can predict the experimentally-observed response of cells treated with latrunculin A, which removes feedback from downstream processes, under a variety of stimulus protocols. We show that Gα2βγ cycling modulated by Ric8, a nonreceptor guanine exchange factor for Gα2 in Dicty, drives multiple phases of Ras activation and leads to direction sensing and signal amplification in cAMP gradients. The model predicts that both Gα2 and Gβγ are essential for direction sensing, in that membrane-localized Gα2*, the activated GTP-bearing form of Gα2, leads to asymmetrical recruitment of RasGEF and Ric8, while globally-diffusing Gβγ mediates their activation. We show that the predicted response at the level of Ras activation encodes sufficient ‘memory’ to eliminate the ‘back-of-the wave’ problem, and the effects of diffusion and cell shape on direction sensing are also investigated. In contrast with existing LEGI models of chemotaxis, the results do not require a disparity between the diffusion coefficients of the Ras activator GEF and the Ras inhibitor GAP. Since the signal pathways we study are highly conserved between Dicty

  1. Control of mRNA stability during development of Dictyostelium discoideum.

    PubMed

    Mangiarotti, G

    1989-01-01

    A large group of mRNA species (which are mainly pre-spore specific) accumulate only after the formation of multicellular aggregates. They are transcribed at a constant rate from the beginning of development and their accumulation is controlled by a 10-20-fold increase in their stability. This mRNA stabilization is dependent upon multicellularity. When aggregates are dispersed, the mRNAs are destabilized; if cells are allowed to reaggregate, the destabilization is reversed. Destabilization is not due to a selective exclusion of mRNA from polyribosomes, but is a primary control event. It does not require synthesis of new RNA or protein, but it may require an interaction between ribosome and the 5'-end of mRNA molecules.

  2. N-glycomic profiling of a glucosidase II mutant of Dictyostelium discoideum by ''off-line'' liquid chromatography and mass spectrometry.

    PubMed

    Hykollari, Alba; Dragosits, Martin; Rendić, Dubravko; Wilson, Iain B H; Paschinger, Katharina

    2014-08-01

    In this study, we have performed the first mass spectrometric analysis of N-glycans of the M31 mutant strain of the cellular slime mould Dictyostelium discoideum, previously shown to have a defect in glucosidase II. Together with glucosidase I, this enzyme mediates part of the initial processing of N-glycans; defects in either glucosidase are associated with human diseases and result in an accumulation of incorrectly processed oligosaccharides which are not, or only poor, substrates for a range of downstream enzymes. To examine the effect of the glucosidase II mutation in Dictyostelium, we employed off-line LC-MALDI-TOF MS in combination with chemical and enzymatic treatments and MS/MS to analyze the neutral and anionic N-glycans of the mutant as compared to the wild type. The major neutral species were, as expected, of the composition Hex10-11 HexNAc2-3 with one or two terminal glucose residues. Consistent with the block in processing of neutral N-glycans caused by the absence of glucosidase II, fucose was apparently absent from the N-glycans and bisecting N-acetylglucosamine was rare. The major anionic oligosaccharides were sulfated and/or methylphosphorylated forms of Hex8-11 HexNAc2-3 , many of which surprisingly lacked glucose residues entirely. As anionic N-glycans are considered to be mostly associated with lysosomal enzymes in Dictyostelium, we hypothesise that glycosidases present in the acidic compartments may act on the oligosaccharides attached to such slime mould proteins. Furthermore, our chosen analytical approach enabled us, via observation of diagnostic negative-mode MS/MS fragments, to determine the fine structure of the methylphosphorylated and sulfated N-glycans of the M31 glucosidase mutant in their native state.

  3. Balanced cortical stiffness is important for efficient migration of Dictyostelium cells in confined environments.

    PubMed

    Roth, Heike; Samereier, Matthias; Trommler, Gudrun; Noegel, Angelika A; Schleicher, Michael; Müller-Taubenberger, Annette

    2015-11-27

    Dictyostelium discoideum cells resemble in many aspects human leukocytes and serve as a model to study actin cytoskeleton dynamics and cell migration of highly motile cells. Dictyostelium cells deficient in the actin-binding protein filamin (ddFLN) showed a surprisingly subtle change in phenotype with no or only minor effects in single cell motility. These findings were in contrast to the strong actin-crosslinking activities measured for filamin in vitro. In the present study, we set out to revisit the role of ddFLN in cell migration. For this purpose, we examined migration of wild-type, ddFLN-null and ddFLN-overexpressing cells under different conditions. In addition to cyclic-AMP chemotaxis assays using micropipettes, we explored cell migration under more confined conditions: an under-agarose 2D assay and a 3D assay employing a collagen matrix that was adapted from assays for leukocytes. Using 3D migration conditions, cells deficient in ddFLN displayed only a minor impairment of motility, similar to the results obtained for migration in 2D. However, cells overexpressing ddFLN showed a remarkable decrease in the speed of migration in particular in 3D environments. We suggest that these results are in line with an increased stiffening of the cortex due to the crosslinking activity of overexpressed ddFLN. Our conclusion is that the absolute level of ddFLN is critical for efficient migration. Furthermore, our results show that under conditions of increased mechanical stress, Dictyostelium cells, like leukocytes, switch to a bleb-based mode of movement.

  4. The locomotion, shape and pseudopodial dynamics of unstimulated Dictyostelium cells are not random.

    PubMed

    Killich, T; Plath, P J; Wei, X; Bultmann, H; Rensing, L; Vicker, M G

    1993-12-01

    The dynamic periphery of unstimulated, preaggregation, hunger-stage Dictyostelium discoideum amoebae was investigated by time-lapse videomicroscopy and digital image processing. Circular maps (i.e. of each of 360 radii around the cell transformed upon Cartesian coordinates) were constructed around the centroid of individual cell images and analysed in time series. This novel technique generated spatiotemporal structures of various degrees of order in the maps, which resemble classical wave interference patterns. The patterns thus demonstrate that cell movement is not random and that cells are intrinsically vibrating bodies, transited by self-organized, superpositioned, harmonic modes of rotating oscillatory waves (ROWS). These waves appear to depend upon spatiotemporal oscillations in the physicochemical reactions associated with actin polymerization, and they govern pseudopodial movements, cell shape and locomotion generally. ROWS in this case are unrelated to the cyclic-AMP-regulated oscillations, which characterize later, aggregative populations of Dictyostelium. However, the exposure of aggregation-stage cells to a pulse of the chemoattractant cyclic-AMP induces a characteristic sequence of changes in the global cellular concentration and spatiotemporal distribution of fibrillar (F-)actin. This reaction begins with what appears to be a phase resetting of ROWS and it may, therefore, underlie the cellular perception of and response to chemotactic signals. We also develop here an analytical mathematical description of ROWS, and use it to simulate cell movements accurately.

  5. UDP-GlcNAc:Glycoprotein N-acetylglucosamine-1-phosphotransferase mediates the initial step in the formation of the methylphosphomannosyl residues on the high mannose oligosaccharides of Dictyostelium discoideum glycoproteins.

    PubMed

    Qian, Yi; West, Christopher M; Kornfeld, Stuart

    2010-03-19

    The Dictyostelium discoideum gene gpt1 encodes a protein XP_638036 with sequence similarity to the alpha/beta subunits of mammalian UDP-GlcNAc:Glycoprotein N-acetylglucosamine-1-phosphotransferase. We now demonstrate that extracts of D. discoideum clones with mutations in this gene transfer GlcNAc-P from UDP-GlcNAc to mannose residues at less than 5% the wild type value. Further, the lysosomal hydrolases of these mutant clones fail to bind to a cation-independent mannose 6-phosphate receptor affinity column, indicating a lack of methylphosphomannosyl residues on the high mannose oligosaccharides of these proteins. We conclude that the gpt1 gene product catalyzes the initial step in the formation of methylphosphomannosyl residues on D. discoideum lysosomal hydrolases.

  6. Control of 6-(D-threo-1',2'-dihydroxypropyl) pterin (dictyopterin) synthesis during aggregation of Dictyostelium discoideum. Involvement of the G-protein-linked signalling pathway in the regulation of GTP cyclohydrolase I activity.

    PubMed Central

    Gütlich, M; Witter, K; Bourdais, J; Veron, M; Rödl, W; Ziegler, I

    1996-01-01

    6-(D-threo-1',2'-Dihydroxypropylpterin (dictyopterin) has been identified in extracts of growing Dictyostelium dicoideum cells [Klein, Thiery and Tatischeff (1990) Eur. J. Biochem. 187, 665-669]. We demonstrate that it originates from GTP by de novo biosynthesis and that the first committed step is catalysed by GTP cyclohydrolase I, yielding dihydroneopterin triphosphate [neopterin is 6-(D-erythro-1',2',3'-trihydroxypropyl) pterin]. The GTP cyclohydrolase I activity is found in the cytosolic fraction and in a membrane-associated form. The level of a 0.9 kb mRNA coding for GTP cyclohydrolase I decreases to about 10% of its initial value within 2 h after Dictyostelium cells start development induced by starvation. In the cytosolic fraction, the specific activities of GTP cyclohydrolase I, as well as the concentrations of (6R/S)-5,6,7,8-tetrahydrodictyopterin (H4dictyopterin), follow this decline of the mRNA level. In the particulate fraction, however, the specific activities of GTP cyclohydrolase I and, in consequence, H4dictyopterin synthesis, transiently increase and reach a maximum after 4-5 h of development. The time-course of H4dictyopterin concentrations in the starvation medium closely correlates with its production in the membrane fraction. The activity of membrane-associated GTP cyclohydrolase I can be increased by pre-incubation of the cell lysate with guanosine 5'-[gamma-thio]triphosphate and Mg2+. This GTP analogue does not serve as a substrate and has no direct effect on the enzyme activity, indicating that a G-protein-linked signalling pathway is involved in the regulation of GTP cyclohydrolase I activity and thus in H4dictyopterin production during early development of D. discoideum. PMID:8660315

  7. Control of 6-(D-threo-1',2'-dihydroxypropyl) pterin (dictyopterin) synthesis during aggregation of Dictyostelium discoideum. Involvement of the G-protein-linked signalling pathway in the regulation of GTP cyclohydrolase I activity.

    PubMed

    Gütlich, M; Witter, K; Bourdais, J; Veron, M; Rödl, W; Ziegler, I

    1996-02-15

    6-(D-threo-1',2'-Dihydroxypropylpterin (dictyopterin) has been identified in extracts of growing Dictyostelium dicoideum cells [Klein, Thiery and Tatischeff (1990) Eur. J. Biochem. 187, 665-669]. We demonstrate that it originates from GTP by de novo biosynthesis and that the first committed step is catalysed by GTP cyclohydrolase I, yielding dihydroneopterin triphosphate [neopterin is 6-(D-erythro-1',2',3'-trihydroxypropyl) pterin]. The GTP cyclohydrolase I activity is found in the cytosolic fraction and in a membrane-associated form. The level of a 0.9 kb mRNA coding for GTP cyclohydrolase I decreases to about 10% of its initial value within 2 h after Dictyostelium cells start development induced by starvation. In the cytosolic fraction, the specific activities of GTP cyclohydrolase I, as well as the concentrations of (6R/S)-5,6,7,8-tetrahydrodictyopterin (H4dictyopterin), follow this decline of the mRNA level. In the particulate fraction, however, the specific activities of GTP cyclohydrolase I and, in consequence, H4dictyopterin synthesis, transiently increase and reach a maximum after 4-5 h of development. The time-course of H4dictyopterin concentrations in the starvation medium closely correlates with its production in the membrane fraction. The activity of membrane-associated GTP cyclohydrolase I can be increased by pre-incubation of the cell lysate with guanosine 5'-[gamma-thio]triphosphate and Mg2+. This GTP analogue does not serve as a substrate and has no direct effect on the enzyme activity, indicating that a G-protein-linked signalling pathway is involved in the regulation of GTP cyclohydrolase I activity and thus in H4dictyopterin production during early development of D. discoideum.

  8. Chemotactic responses of Dictyostelium discoideum amoebae to a cyclic AMP concentration gradient: evidence to support a spatial mechanism for sensing cyclic AMP.

    PubMed

    Tani, T; Naitoh, Y

    1999-01-01

    The motile responses of Dictyostelium discoideum amoebae to a cyclic AMP (cAMP) concentration gradient were examined using a novel assay system. In this system, a cAMP concentration gradient was generated, while the overall cAMP concentration could be either increased or decreased in a chamber containing amoebae. The chemotactic responses of amoebae were examined immediately after they had been subjected to the cAMP concentration gradient. Amoebae moving in random directions in a reference solution ascended a cAMP concentration gradient after they had been exposed to the gradient irrespective of whether there was an increase or a decrease in the overall cAMP concentration. This strongly supports the idea that D. discoideum amoebae can sense a spatial cAMP gradient around them and that this causes their chemoaccumulation behavior. Ascending locomotion became less conspicuous when the amoebae were treated with a homogeneous cAMP solution for approximately 8 min before exposure to a cAMP gradient. This cAMP pretreatment reduced the sensitivity of the amoeba to a cAMP concentration gradient. The cAMP concentration gradient could be reversed in less than 30 s in this assay system, allowing the generation of a cAMP wave by accumulating amoebae to be mimicked. The ascending amoebae continued to move in the same direction for 1-2 min after the gradient had been reversed. This is consistent with the well-known observation that reversal of a cAMP concentration gradient experienced by the amoebae passing through a cAMP wave does not negate their chemotactic movement towards the accumulation center.

  9. Determinants of mRNA stability in Dictyostelium discoideum amoebae: differences in poly(A) tail length, ribosome loading, and mRNA size cannot account for the heterogeneity of mRNA decay rates.

    PubMed Central

    Shapiro, R A; Herrick, D; Manrow, R E; Blinder, D; Jacobson, A

    1988-01-01

    As an approach to understanding the structures and mechanisms which determine mRNA decay rates, we have cloned and begun to characterize cDNAs which encode mRNAs representative of the stability extremes in the poly(A)+ RNA population of Dictyostelium discoideum amoebae. The cDNA clones were identified in a screening procedure which was based on the occurrence of poly(A) shortening during mRNA aging. mRNA half-lives were determined by hybridization of poly(A)+ RNA, isolated from cells labeled in a 32PO4 pulse-chase, to dots of excess cloned DNA. Individual mRNAs decayed with unique first-order decay rates ranging from 0.9 to 9.6 h, indicating that the complex decay kinetics of total poly(A)+ RNA in D. discoideum amoebae reflect the sum of the decay rates of individual mRNAs. Using specific probes derived from these cDNA clones, we have compared the sizes, extents of ribosome loading, and poly(A) tail lengths of stable, moderately stable, and unstable mRNAs. We found (i) no correlation between mRNA size and decay rate; (ii) no significant difference in the number of ribosomes per unit length of stable versus unstable mRNAs, and (iii) a general inverse relationship between mRNA decay rates and poly(A) tail lengths. Collectively, these observations indicate that mRNA decay in D. discoideum amoebae cannot be explained in terms of random nucleolytic events. The possibility that specific 3'-structural determinants can confer mRNA instability is suggested by a comparison of the labeling and turnover kinetics of different actin mRNAs. A correlation was observed between the steady-state percentage of a given mRNA found in polysomes and its degree of instability; i.e., unstable mRNAs were more efficiently recruited into polysomes than stable mRNAs. Since stable mRNAs are, on average, "older" than unstable mRNAs, this correlation may reflect a translational role for mRNA modifications that change in a time-dependent manner. Our previous studies have demonstrated both a time

  10. Genetic control of morphogenesis in Dictyostelium

    PubMed Central

    Loomis, William F.

    2015-01-01

    Cells grow, move, expand, shrink and die in the process of generating the characteristic shapes of organisms. Although the structures generated during development of the social amoeba Dictyostelium discoideum look nothing like the structures seen in metazoan embryogenesis, some of the morphogenetic processes used in their making are surprisingly similar. Recent advances in understanding the molecular basis for directed cell migration, cell type specific sorting, differential adhesion, secretion of matrix components, pattern formation, regulation and terminal differentiation are reviewed. Genes involved in Dictyostelium aggregation, slug formation, and culmination of fruiting bodies are discussed. PMID:25872182

  11. Genetic control of morphogenesis in Dictyostelium.

    PubMed

    Loomis, William F

    2015-06-15

    Cells grow, move, expand, shrink and die in the process of generating the characteristic shapes of organisms. Although the structures generated during development of the social amoeba Dictyostelium discoideum look nothing like the structures seen in metazoan embryogenesis, some of the morphogenetic processes used in their making are surprisingly similar. Recent advances in understanding the molecular basis for directed cell migration, cell type specific sorting, differential adhesion, secretion of matrix components, pattern formation, regulation and terminal differentiation are reviewed. Genes involved in Dictyostelium aggregation, slug formation, and culmination of fruiting bodies are discussed.

  12. Quantitation of Membrane Sites in Aggregating Dictyostelium Cells by Use of Tritiated Univalent Antibody

    PubMed Central

    Beug, H.; Katz, F. E.; Stein, A.; Gerisch, G.

    1973-01-01

    Cell-to-cell adhesion during aggregation of Dictyostelium discoideum cells is completely blocked by univalent antibody (Fab) directed against two classes of target sites: surface structures characteristic for aggregation-competent cells (“contact sites A”) and others present also on growth-phase cells (“contact sites B”). 3 × 105 Fab molecules bound per cell are sufficient to block contact sites A completely, although the Fab fragments cover not more than 2% of the total cell surface. Up to 8-fold this value can be bound per cell when Fab fragments of another specificity are used, without affecting activity of contact sites A. Blockage of cell-to-cell adhesion therefore depends on the binding of Fab fragments to specific target sites, rather than on the total number of Fab molecules bound per cell. This conclusion is also valid for cell adhesion attributed to contact sites B. Contact sites therefore represent a special class of cell-surface sites which, in cell homogenates as well as in vivo, can be traced by Fab, and which are not identical with the bulk of cell-surface antigens present on aggregating cells. Images PMID:4522296

  13. Biochemical Responses to Chemically Distinct Chemoattractants During the Growth and Development of Dictyostelium.

    PubMed

    Meena, Netra Pal; Kimmel, Alan R

    2016-01-01

    Dictyostelium discoideum has proven an excellent model for the study of eukaryotic chemotaxis. During growth in its native environment, Dictyostelium phagocytose bacteria and fungi for primary nutrient capture. Growing Dictyostelium can detect these nutrient sources through chemotaxis toward the metabolic by-product folate. Although Dictyostelium grow as individual cells, nutrient depletion induces a multicellular development program and a separate chemotactic response pathway. During development, Dictyostelium synthesize and secrete cAMP, which serves as a chemoattractant to mobilize and coordinate cells for multicellular formation and development. Separate classes of GPCRs and Gα proteins mediate chemotactic signaling to the chemically distinct ligands. We discuss common and separate component responses of Dictyostelium to folate and cAMP during growth and development, and the advantages and disadvantages for each. As examples, we present biochemical assays to characterize the chemoattractant-induced kinase activations of mTORC2 and the ERKs.

  14. Role of SpdA in Cell Spreading and Phagocytosis in Dictyostelium

    PubMed Central

    Dias, Marco; Brochetta, Cristiana; Marchetti, Anna; Bodinier, Romain; Brückert, Franz; Cosson, Pierre

    2016-01-01

    Dictyostelium discoideum is a widely used model to study molecular mechanisms controlling cell adhesion, cell spreading on a surface, and phagocytosis. In this study we isolated and characterize a new mutant created by insertion of a mutagenic vector in the heretofore uncharacterized spdA gene. SpdA-ins mutant cells produce an altered, slightly shortened version of the SpdA protein. They spread more efficiently than WT cells when allowed to adhere to a glass substrate, and phagocytose particles more efficiently. On the contrary, a functional spdA knockout mutant where a large segment of the gene was deleted phagocytosed less efficiently and spread less efficiently on a substrate. These phenotypes were highly dependent on the cellular density, and were most visible at high cell densities, where secreted quorum-sensing factors inhibiting cell motility, spreading and phagocytosis are most active. These results identify the involvement of SpdA in the control of cell spreading and phagocytosis. The underlying molecular mechanisms, as well as the exact link between SpdA and cell spreading, remain to be established. PMID:27512991

  15. A new spore differentiation factor (SDF) secreted by Dictyostelium cells is phosphorylated by the cAMP dependent protein kinase.

    PubMed

    Anjard, C; van Bemmelen, M; Véron, M; Reymond, C D

    1997-10-01

    Upon starvation, Dictyostelium discoideum unicellular amoebae form a multicellular organism leading to the development of a fruiting body containing spores. Single cells of sporogenous mutants, unlike wild type cells, are able to differentiate into spores under specific conditions. We show in this report that overexpression of the catalytic subunit of the cAMP dependent protein kinase (PKA), not only renders the cells sporogenous, but is also accompanied by the production/release of a diffusible spore differentiation factor (SDF). SDF is a small, thermostable phospho-polypeptide. In vitro dephosphorylation reduces SDF spore differentiation capacity, which can be regained in vitro by PKA phosphorylation. These results indicate that SDF is a PKA substrate and might be activated in vivo by this protein kinase. Since spore differentiation requires PKA catalytic subunit activation, we conclude that the response of prespore cells to SDF involves an intracellular pathway dependent on PKA.

  16. Dictyostelium mutants lacking the cytoskeletal protein coronin are defective in cytokinesis and cell motility

    PubMed Central

    1993-01-01

    Coronin is an actin-binding protein in Dictyostelium discoideum that is enriched at the leading edge of the cells and in projections of the cell surface called crowns. The polypeptide sequence of coronin is distinguished by its similarities to the beta-subunits of trimeric G proteins (E. L. de Hostos, B. Bradtke, F. Lottspeich, R. Guggenheim, and G. Gerisch, 1991. EMBO (Eur. Mol. Biol. Organ.) J. 10:4097-4104). To elucidate the in vivo function of coronin, null mutants have been generated by gene replacement. The mutant cells lacking coronin grow and migrate more slowly than wild-type cells. When these cor- cells grow in liquid medium they become multinucleate, indicating a role of coronin in cytokinesis. To explore this role, coronin has been localized in mitotic wild-type cells by immunofluorescence labeling. During separation of the daughter cells, coronin is strongly accumulated at their distal portions including the leading edges. This contrasts with the localization of myosin II in the cleavage furrow and suggests that coronin functions independently of the conventional myosin in facilitating cytokinesis. PMID:8380174

  17. Inference of the drivers of collective movement in two cell types: Dictyostelium and melanoma

    PubMed Central

    Matthiopoulos, Jason; Husmeier, Dirk

    2016-01-01

    Collective cell movement is a key component of many important biological processes, including wound healing, the immune response and the spread of cancers. To understand and influence these movements, we need to be able to identify and quantify the contribution of their different underlying mechanisms. Here, we define a set of six candidate models—formulated as advection–diffusion–reaction partial differential equations—that incorporate a range of cell movement drivers. We fitted these models to movement assay data from two different cell types: Dictyostelium discoideum and human melanoma. Model comparison using widely applicable information criterion suggested that movement in both of our study systems was driven primarily by a self-generated gradient in the concentration of a depletable chemical in the cells' environment. For melanoma, there was also evidence that overcrowding influenced movement. These applications of model inference to determine the most likely drivers of cell movement indicate that such statistical techniques have potential to support targeted experimental work in increasing our understanding of collective cell movement in a range of systems. PMID:27798280

  18. Abnormalities of Endocytosis, Phagocytosis, and Development Process in Dictyostelium Cells That Over-Express Acanthamoeba castellanii Metacaspase Protein

    PubMed Central

    SAHEB, Entsar; TRZYNA, Wendy; MARINGER, Katherine; BUSH, John

    2015-01-01

    Background: Acanthamoeba castellanii forms a resistant cyst that protects the parasite against the host’s immune response. Acanthamoeba Type-I metacaspase (Acmcp) is a caspase-like protein that has been found to be expressed during the encystations. Dictyostelium discoideum is an organism closely related to Acanthamoeba useful for studying the molecular function of this protozoan caspase-like protein. Methods: The full length of Acmcp and a mutated version of the same gene, which lacks the proline rich N-terminal region (Acmcp-dpr), were cloned into the pDneo2a-GFP vector separately. The pDneo2a-GFP-Acmcp and pDneo2a-GFPAcmcp-dpr were electro-transfected into wild type D. discoideum cells to create cell lines that over-expressed Acmcp or Acmcp-dpr. Results: Both cell lines that over-expressed Acmcp and Acmcp-dpr showed a significant increase in the fluid phase internalization and phagocytosis rate compared to the control cells. Additionally, the cells expressing the Acmcp-dpr mutant were unable to initiate early development and failed to aggregate or form fruiting bodies under starvation conditions, whereas Acmcp over-expressing cells showed the opposite phenomena. Quantitative cell death analysis provided additional support for these findings. Conclusion: Acmcp is involved in the processes of endocytosis and phagocytosis. In addition, the proline rich region in Acmcp is important for cellular development in Dictyostelium. Given its important role in the development process, metacaspase protein is proposed as a candidate drug target against infections caused by A. castellanii. PMID:26246819

  19. A homologue of Cdk8 is required for spore cell differentiation in Dictyostelium.

    PubMed

    Lin, Hsiu-Hsu Sophia; Khosla, Meenal; Huang, Hao-Jen; Hsu, Duen-Wei; Michaelis, Christine; Weeks, Gerald; Pears, Catherine

    2004-07-01

    The Cdk8 proteins are kinases which phosphorylate the carboxy terminal domain (CTD) of RNA polymerase II (Pol II) as well as some transcription factors and, therefore, are involved in the regulation of transcription. Here, we report that a Cdk8 homologue from Dictyostelium discoideum is localized in the nucleus where it forms part of a high molecular weight complex that has CTD kinase activity. Insertional mutagenesis was used to abrogate gene function, and analysis of the null strain revealed that the DdCdk8 protein plays an important role in spore formation during late development. As previously reported [Dev. Growth Differ. 44 (2002) 213] Ddcdk8- cells also exhibit impaired aggregation, although we report that the severity of the defect depends upon experimental conditions. When aggregation occurs, Ddcdk8- cells form abnormal terminally differentiated structures within which the Ddcdk8- cells differentiate into stalk cells but fail to form spores, indicating a role for DdCdk8 in cell differentiation. When Ddcdk8 is expressed from its own promoter, the protein is able to rescue both the late developmental defect and the impaired aggregation. However, when expressed from an heterologous promoter, only the impaired aggregation is rescued. This result demonstrates that the defect during late development is not a consequence of impaired aggregation and indicates a direct role for DdCdk8 in spore formation.

  20. Reaction-diffusion waves of actin filament polymerization/depolymerization in Dictyostelium pseudopodium extension and cell locomotion.

    PubMed

    Vicker, M G

    2000-04-14

    Cell surface movements and the intracellular spatial patterns and dynamics of actin filament (F-actin) were investigated in living and formalin-fixed cells of Dictyostelium discoideum by confocal microscopy. Excitation waves of F-actin assembly developed and propagated several micrometers at up to 26 microm/min in cells which had been intracellularly loaded with fluorescently labeled actin monomer. Wave propagation and extinction corresponded with the initiation and attenuation of pseudopodium extension and cell advance, respectively. The identification of chemical waves was supported by the ring, sphere, spiral and scroll wave patterns, which were observed in the extensions of fixed cells stained with phalloidin-rhodamine, and by the similar, asymmetrical [F-actin] distribution in wavefronts in living and fixed cells. These F-actin patterns and dynamics in Dictyostelium provide evidence for a new supramolecular state of actin, which propagates as a self-organized, reaction-diffusion wave of reversible F-actin assembly and affects pseudopodium extension. Actin's properties of oscillation and self-organization might also fundamentally determine the nature of the eukaryotic cell's reactions of adaptation, timing and signal response.

  1. An Experimental System to Study Cell Differentiation.

    ERIC Educational Resources Information Center

    Bozzone, Donna M.

    1994-01-01

    Uses Dictyostelium discoideum to aid in introducing cell differentiation to students. Students engage in a laboratory exercise that allows them to investigate the means by which embryonic cells choose developmental pathways. (ZWH)

  2. Desynchronization of Cells on the Developmental Path Triggers the Formation of Spiral Waves of cAMP during Dictyostelium Aggregation

    NASA Astrophysics Data System (ADS)

    Lauzeral, Jacques; Halloy, Jose; Goldbeter, Albert

    1997-08-01

    Whereas it is relatively easy to account for the formation of concentric (target) waves of cAMP in the course of Dictyostelium discoideum aggregation after starvation, the origin of spiral waves remains obscure. We investigate a physiologically plausible mechanism for the spontaneous formation of spiral waves of cAMP in D. discoideum. The scenario relies on the developmental path associated with the continuous changes in the activity of enzymes such as adenylate cyclase and phosphodiesterase observed during the hours that follow starvation. These changes bring the cells successively from a nonexcitable state to an excitable state in which they relay suprathreshold cAMP pulses, and then to autonomous oscillations of cAMP, before the system returns to an excitable state. By analyzing a model for cAMP signaling based on receptor desensitization, we show that the desynchronization of cells on this developmental path triggers the formation of fully developed spirals of cAMP. Developmental paths that do not correspond to the sequence of dynamic transitions no relay-relay-oscillations-relay are less able or fail to give rise to the formation of spirals.

  3. Extracellular chemical signal controlling phototactic behavior by D. discoideum slugs

    SciTech Connect

    Fisher, P.R.; Smith, E.; Williams, K.L.

    1981-03-01

    Developing cells of the cellular slime mold Dictyostelium discoideum release a low molecular weight metabolite (Slug Turning Factor, STF) which, at high uniform concentrations, interferes with phototaxis and thermotaxis by D. discoideum slugs. D. discoideum slugs migrating in darkness are repelled by (exhibit negative chemotaxis to) crude STF exudates. Dose-response curves relating the accuracies of phototaxis and negative chemotaxis to STF concentration indicate that, in both phototaxis and chemotaxis, slugs measure the ratios of STF concentrations on their opposite sides. Net STF release is enhanced by light. Researchers propose that light, focused onto the slug's distal side by its convex surface, generates a lateral STF gradient in response to which the slug turns toward the light source.

  4. The Dictyostelium discoideum RNA-dependent RNA polymerase RrpC silences the centromeric retrotransposon DIRS-1 post-transcriptionally and is required for the spreading of RNA silencing signals

    PubMed Central

    Wiegand, Stephan; Meier, Doreen; Seehafer, Carsten; Malicki, Marek; Hofmann, Patrick; Schmith, Anika; Winckler, Thomas; Földesi, Balint; Boesler, Benjamin; Nellen, Wolfgang; Reimegård, Johan; Käller, Max; Hällman, Jimmie; Emanuelsson, Olof; Avesson, Lotta; Söderbom, Fredrik; Hammann, Christian

    2014-01-01

    Dictyostelium intermediate repeat sequence 1 (DIRS-1) is the founding member of a poorly characterized class of retrotransposable elements that contain inverse long terminal repeats and tyrosine recombinase instead of DDE-type integrase enzymes. In Dictyostelium discoideum, DIRS-1 forms clusters that adopt the function of centromeres, rendering tight retrotransposition control critical to maintaining chromosome integrity. We report that in deletion strains of the RNA-dependent RNA polymerase RrpC, full-length and shorter DIRS-1 messenger RNAs are strongly enriched. Shorter versions of a hitherto unknown long non-coding RNA in DIRS-1 antisense orientation are also enriched in rrpC– strains. Concurrent with the accumulation of long transcripts, the vast majority of small (21 mer) DIRS-1 RNAs vanish in rrpC– strains. RNASeq reveals an asymmetric distribution of the DIRS-1 small RNAs, both along DIRS-1 and with respect to sense and antisense orientation. We show that RrpC is required for post-transcriptional DIRS-1 silencing and also for spreading of RNA silencing signals. Finally, DIRS-1 mis-regulation in the absence of RrpC leads to retrotransposon mobilization. In summary, our data reveal RrpC as a key player in the silencing of centromeric retrotransposon DIRS-1. RrpC acts at the post-transcriptional level and is involved in spreading of RNA silencing signals, both in the 5′ and 3′ directions. PMID:24369430

  5. TipC and the chorea-acanthocytosis protein VPS13A regulate autophagy in Dictyostelium and human HeLa cells.

    PubMed

    Muñoz-Braceras, Sandra; Calvo, Rosa; Escalante, Ricardo

    2015-01-01

    Deficient autophagy causes a distinct phenotype in Dictyostelium discoideum, characterized by the formation of multitips at the mound stage. This led us to analyze autophagy in a number of multitipped mutants described previously (tipA(-), tipB(-), tipC(-), and tipD(-)). We found a clear autophagic dysfunction in tipC(-) and tipD(-) while the others showed no defects. tipD codes for a homolog of Atg16, which confirms the role of this protein in Dictyostelium autophagy and validates our approach. The tipC-encoded protein is highly similar to human VPS13A (also known as chorein), whose mutations cause the chorea-acanthocytosis syndrome. No member of the VPS13 protein family has been previously related to autophagy despite the presence of a region of similarity to Atg2 at the C terminus. This region also contains the conserved domain of unknown function DUF1162. Of interest, the expression of the TipC C-terminal coding sequence containing these 2 motifs largely complemented the mutant phenotype. Dictyostelium cells lacking TipC displayed a reduced number of autophagosomes visualized with the markers GFP-Atg18 and GFP-Atg8 and an impaired autophagic degradation as determined by a proteolytic cleavage assay. Downregulation of human VPS13A in HeLa cells by RNA interference confirmed the participation of the human protein in autophagy. VPS13A-depleted cells showed accumulation of autophagic markers and impaired autophagic flux.

  6. Derivatives of Dictyostelium differentiation-inducing factors inhibit lysophosphatidic acid–stimulated migration of murine osteosarcoma LM8 cells

    SciTech Connect

    Kubohara, Yuzuru; Komachi, Mayumi; Homma, Yoshimi; Kikuchi, Haruhisa; Oshima, Yoshiteru

    2015-08-07

    Osteosarcoma is a common metastatic bone cancer that predominantly develops in children and adolescents. Metastatic osteosarcoma remains associated with a poor prognosis; therefore, more effective anti-metastatic drugs are needed. Differentiation-inducing factor-1 (DIF-1), −2, and −3 are novel lead anti-tumor agents that were originally isolated from the cellular slime mold Dictyostelium discoideum. Here we investigated the effects of a panel of DIF derivatives on lysophosphatidic acid (LPA)-induced migration of mouse osteosarcoma LM8 cells by using a Boyden chamber assay. Some DIF derivatives such as Br-DIF-1, DIF-3(+2), and Bu-DIF-3 (5–20 μM) dose-dependently suppressed LPA-induced cell migration with associated IC{sub 50} values of 5.5, 4.6, and 4.2 μM, respectively. On the other hand, the IC{sub 50} values of Br-DIF-1, DIF-3(+2), and Bu-DIF-3 versus cell proliferation were 18.5, 7.2, and 2.0 μM, respectively, in LM8 cells, and >20, 14.8, and 4.3 μM, respectively, in mouse 3T3-L1 fibroblasts (non-transformed). Together, our results demonstrate that Br-DIF-1 in particular may be a valuable tool for the analysis of cancer cell migration, and that DIF derivatives such as DIF-3(+2) and Bu-DIF-3 are promising lead anti-tumor agents for the development of therapies that suppress osteosarcoma cell proliferation, migration, and metastasis. - Highlights: • LPA induces cell migration (invasion) in murine osteosarcoma LM8 cells. • DIFs are novel lead anti-tumor agents found in Dictyostelium discoideum. • We examined the effects of DIF derivatives on LPA-induced LM8 cell migration in vitro. • Some of the DIF derivatives inhibited LPA-induced LM8 cell migration.

  7. Mound-cell movement and morphogenesis in Dictyostelium.

    PubMed

    Kellerman, K A; McNally, J G

    1999-04-15

    To examine the mechanisms of cell locomotion within a three-dimensional (3-D) cell mass, we have undertaken a systematic 3-D analysis of individual cell movements in the Dictyostelium mound, the first 3-D structure to form during development of the fruiting body. We used time-lapse deconvolution microscopy to examine two strains whose motion represents endpoints on the spectrum of motile behaviors that we have observed in mounds. In AX-2 mounds, cell motion is slow and trajectories are a combination of random and radial, compared to KAX-3, in which motion is fivefold faster and most trajectories are rotational. Although radial or rotational motion was correlated with the optical-density wave patterns present in each strain, we also found small but significant subpopulations of cells that moved differently from the majority, demonstrating that optical-density waves are at best insufficient to explain all motile behavior in mounds. In examining morphogenesis in these strains, we noted that AX-2 mounds tended to culminate directly to a fruiting body, whereas KAX-3 mounds first formed a migratory slug. By altering buffering conditions we could interchange these behaviors and then found that mound-cell motions also changed accordingly. This demonstrates a correlation between mound-cell motion and subsequent development, but it is not obligatory. Chimeric mounds composed of only 10% KAX-3 cells and 90% AX-2 cells exhibited rotational motion, suggesting that a diffusible molecule induces rotation, but many of these mounds still culminated directly, demonstrating that rotational motion does not always lead to slug migration. Our observations provide a detailed analysis of cell motion for two distinct modes of mound and slug formation in Dictyostelium.

  8. Talin-Null Cells of Dictyostelium Are Strongly Defective in Adhesion to Particle and Substrate Surfaces and Slightly Impaired in Cytokinesis

    PubMed Central

    Niewöhner, Jens; Weber, Igor; Maniak, Markus; Müller-Taubenberger, Annette; Gerisch, Günther

    1997-01-01

    Dictyostelium discoideum contains a full-length homologue of talin, a protein implicated in linkage of the actin system to sites of cell-to-substrate adhesion in fibroblasts and neuronal growth cones. Gene replacement eliminated the talin homologue in Dictyostelium and led to defects in phagocytosis and cell-to-substrate interaction of moving cells, two processes dependent on a continuous cross talk between the cell surface and underlying cytoskeleton. The uptake rate of yeast particles was reduced, and only bacteria devoid of the carbohydrate moiety of cell surface lipopolysaccharides were adhesive enough to be recruited by talin-null cells in suspension and phagocytosed. Cell-to-cell adhesion of undeveloped cells was strongly impaired in the absence of talin, in contrast with the cohesion of aggregating cells mediated by the phospholipid-anchored contact site A glycoprotein, which proved to be less talin dependent. The mutant cells were still capable of moving and responding to a chemoattractant, although they attached only loosely to a substrate via small areas of their surface. With their high proportion of binucleated cells, the talin-null mutants revealed interactions of the mitotic apparatus with the cell cortex that were not obvious in mononucleated cells. PMID:9230077

  9. Cell movement and shape are non-random and determined by intracellular, oscillatory rotating waves in Dictyostelium amoebae.

    PubMed

    Killich, T; Plath, P J; Hass, E C; Xiang, W; Bultmann, H; Rensing, L; Vicker, M G

    1994-01-01

    We present evidence for a mechanism of eukaryotic cell movement. The pseudopodial dynamics and shape of Dictyostelium discoideum amoebae were investigated using computer-supported video microscopy. An examination of the cell periphery by the novel method of serial circular maps revealed explicit, classical wave patterns, which indicate the existence of intrinsic intracellular oscillations. The patterns are generated by the transit of self-organized, super-positioned, harmonic modes of rotating oscillatory waves (ROWS). These waves are probably associated with the dynamics of intracellular actin polymerisation and depolymerisation. A Karhunen-Loève expansion was conducted on one cell during 10 min of locomotion using points each 10 degrees around the cell's boundary. The results show that only 2-3 modes are necessary to describe the most essential features of cell movement and shape. Based on this analysis, a wave model was developed, which accurately simulates the dynamics of cell movement and shape during this time. The model was tested by reconstructing the cell's dynamical form by means of the Karhunen-Loève transform. No difference was detected between this reconstruction and the actual cell outline. Although cell movement and shape have hitherto been viewed as random, our results demonstrate that ROWS determine the spatio-temporal expression of pseudopodia, and consequently govern cell shape and movement, non-randomly.

  10. Ligand-induced changes in the location of actin, myosin, 95K (alpha- actinin), and 120K protein in amebae of Dictyostelium discoideum

    PubMed Central

    1985-01-01

    In this study we investigated concanavalin A (Con A) induced changes in the locations of actin, myosin, 120K, and 95K (alpha-actinin) to determine the extent to which actin and myosin are reorganized during capping and the roles that 120K and 95K might play in this reorganization. We observed the location of each protein by indirect immunofluorescence using affinity purified antibodies. Four morphological states were distinguished in vegetative Dictyostelium amebae: ameboid cells before Con A binding, patched cells, capped cells, and ameboid cells with caps. The location of each protein was distinct in ameboid cells both before and after capping Actin and 120K were found in the cell cortex usually associated with surface projections, and myosin and 95K were diffusely distributed. Myosin was excluded from surface projections in ameboid cells. During patching, all four proteins were localized below Con A patches. During capping, actin, myosin, and 95K protein moved with the Con A patches into the cap whereas 120K protein was excluded from the cap. During the late stages of cap formation actin and myosin were progressively lost from the cap, and 120K became concentrated in new actin-filled projections that formed away from the cap. However, 95K remained tightly associated with the cap. Poisoning cells with sodium azide inhibited capping but not patching of ligand. In azide-poisoned cells, myosin and 95K did not co-patch with Con A, whereas copatching of 120K and actin with Con A occurred as usual. Our results support the hypothesis that capping is an actomyosin-mediated motile event that involves a sliding interaction between actin filaments, which are anchored through the membrane to ligand patches, and myosin in the cortex. They are also consistent with a role for 120K in the formation of surface projections by promoting growth and/or cross-linking of actin filaments within projections, and with a role for 95K in regulating actomyosin-mediated contractility, earlier

  11. The signal to move: D. discoideum go orienteering.

    PubMed

    Kimmel, Alan R; Parent, Carole A

    2003-06-06

    Cells migrating directionally toward a chemoattractant source display a highly polarized cytoskeletal organization, with F-actin localized predominantly at the anterior and myosin II at the lateral and posterior regions. Dictyostelium discoideum has proven a useful system for elucidating signaling pathways that regulate this chemotactic response. During development, extracellular adenosine 3', 5' monophosphate (cAMP) functions as a primary signal to activate cell surface cAMP receptors (cARs). These receptors transduce different signals depending on whether or not they are coupled to heterotrimeric guanine nucleotide-binding proteins (G proteins) (see the STKE Connections Maps). Multiple G protein-stimulated pathways interact to establish polarity in chemotaxing D. discoideum cells by localizing F-actin at their leading edge and by regulating the phosphorylation state and assembly of myosin II. Many of the molecular interactions described are fundamental to the regulation of chemotaxis in other eukaryotic cells.

  12. Excitability in Dictyostelium development

    NASA Astrophysics Data System (ADS)

    Schwab, David

    2013-03-01

    Discovering how populations of cells reliably develop into complex multi-cellular structures is a key challenge in modern developmental biology. This requires an understanding of how networks at the single-cell level, when combined with intercellular signaling and environmental cues, give rise to the collective behaviors observed in cellular populations. I will present work in collaboration with the Gregor lab, showing that the signal-relay response of starved cells of the amoebae Dictyostelium discoideum can be well modeled as an excitable system. This is in contrast to existing models of the network that postulate a feed-forward cascade. I then extend the signal-relay model to describe how spatial gradient sensing may be achieved via excitability. One potential advantage of relying on feedback for gradient sensing is in preventing ``cheaters'' that do not produce signals from taking over the population. I then combine these models of single-cell signaling and chemotaxis to perform large-scale agent-based simulations of aggregating populations. This allows direct study of how variations in single-cell dynamics modify population behavior. In order to further test this model, I use the results of a screen for mutant cell lines that exhibit altered collective patterns. Finally, I use an existing FRET movie database of starved cell populations at varying cell densities and dilution rates to study heterogeneity in repeated spatio-temporal activity patterns.

  13. LagC is required for cell-cell interactions that are essential for cell-type differentiation in Dictyostelium.

    PubMed

    Dynes, J L; Clark, A M; Shaulsky, G; Kuspa, A; Loomis, W F; Firtel, R A

    1994-04-15

    Strain AK127 is a developmental mutant of Dictyostelium discoideum that was isolated by restriction enzyme-mediated integration (REMI). Mutant cells aggregate normally but are unable to proceed past the loose aggregate stage. The cloned gene, lagC (loose aggregate C), encodes a novel protein of 98 kD that contains an amino-terminal signal sequence and a putative carboxy-terminal transmembrane domain. The mutant strain AK127 shows no detectable lagC transcript upon Northern analysis, indicating that the observed phenotype is that of a null allele. Expression of the lagC cDNA in AK127 cells complements the arrest at the loose aggregate stage, indicating that the mutant phenotype results from disruption of the lagC gene. In wild-type cells, lagC mRNA is induced at the loose aggregate stage and is expressed through the remainder of development. lagC- null cells aggregate but then disaggregate and reaggregate to form small granular mounds. Mature spores are produced at an extremely low efficiency (< 0.1% of wild type), appearing only after approximately 72 hr, whereas wild-type strains produce mature spores by 26 hr. lagC- null cells accumulate reduced levels of transcripts for the prestalk-enriched genes rasD and CP2 and do not express the DIF-induced prestalk-specific gene ecmA or the cAMP-induced prespore-specific gene SP60 to significant levels. In chimeric organisms resulting from the coaggregation of lagC- null and wild-type cells, cell-type-specific gene expression is rescued in the lagC- null cells; however, lagC- prespore cells are localized to the posterior of the prespore region and do not form mature spores, suggesting that LagC protein has both no cell-autonomous and cell-autonomous functions. Overexpression of lagC from an actin promoter in both wild-type and lagC- cells causes a delay at the tight aggregate stage, the first stage requiring LagC activity. These results suggest that the LagC protein functions as a nondiffusible cell-cell signaling molecule

  14. Amebae of Dictyostelium discoideum respond to an increasing temporal gradient of the chemoattractant cAMP with a reduced frequency of turning: evidence for a temporal mechanism in ameboid chemotaxis.

    PubMed

    Varnum-Finney, B; Edwards, K B; Voss, E; Soll, D R

    1987-01-01

    In an aggregation territory of Dictyostelium discoideum, outwardly moving, nondissipating waves of the chemoattractant cAMP sweep across each ameba. At the front of each wave, an ameba experiences an increasing temporal and a positive spatial gradient of cAMP. At the back of a wave, an ameba experiences a decreasing temporal and a negative spatial gradient of cAMP. Employing a perfusion chamber, we have mimicked the temporal dynamics of these waves in the absence of a spatial gradient and demonstrated that the frequency of lateral pseudopod formation and the frequency of turning are dramatically affected by the direction and dynamics of the temporal gradient. In addition, since an ameba will move in a directed fashion up a shallow, nonpulsatile gradient of cAMP, we also mimicked the increasing temporal gradient generated by an ameba moving up a shallow spatial gradient. The frequency of lateral pseudopod formation and the frequency of turning were depressed. Together, these results demonstrate that amebae can assess the direction of a temporal gradient of chemoattractant in the absence of a spatial gradient and alter both the frequency of pseudopod extension and turning, accordingly. Although these results do not rule out the involvement of a spatial mechanism in assessing a spatial gradient, they strongly suggest that the temporal dynamics of a cAMP wave or the temporal gradient generated by an ameba moving through a spatial gradient may play a major role in chemotaxis.

  15. Guenther Gerisch and Dictyostelium, the microbial model for ameboid motility and multicellular morphogenesis.

    PubMed

    Bozzaro, Salvatore; Fisher, Paul R; Loomis, William; Satir, Peter; Segall, Jeffrey E

    2004-10-01

    Beginning in 1960 and continuing to this day, Guenther Gerisch's work on the social ameba Dictyostelium discoideum has helped to make it the model organism of choice for studies of cellular activities that depend upon the actomyosin cytoskeleton. Gerisch has brought insight and quantitative rigor to cell biology by developing novel assays and by applying advanced genetic, biochemical and microscopic techniques to topics as varied as cell-cell adhesion, chemotaxis, motility, endocytosis and cytokinesis.

  16. Ras proteins have multiple functions in vegetative cells of Dictyostelium.

    PubMed

    Bolourani, Parvin; Spiegelman, George; Weeks, Gerald

    2010-11-01

    During the aggregation of Dictyostelium cells, signaling through RasG is more important in regulating cyclic AMP (cAMP) chemotaxis, whereas signaling through RasC is more important in regulating the cAMP relay. However, RasC is capable of substituting for RasG for chemotaxis, since rasG⁻ cells are only partially deficient in chemotaxis, whereas rasC⁻/rasG⁻ cells are totally incapable of chemotaxis. In this study we have examined the possible functional overlap between RasG and RasC in vegetative cells by comparing the vegetative cell properties of rasG⁻, rasC⁻, and rasC⁻/rasG⁻ cells. In addition, since RasD, a protein not normally found in vegetative cells, is expressed in vegetative rasG⁻ and rasC⁻/rasG⁻ cells and appears to partially compensate for the absence of RasG, we have also examined the possible functional overlap between RasG and RasD by comparing the properties of rasG⁻ and rasC⁻/rasG⁻ cells with those of the mutant cells expressing higher levels of RasD. The results of these two lines of investigation show that RasD is capable of totally substituting for RasG for cytokinesis and growth in suspension, whereas RasC is without effect. In contrast, for chemotaxis to folate, RasC is capable of partially substituting for RasG, but RasD is totally without effect. Finally, neither RasC nor RasD is able to substitute for the role that RasG plays in regulating actin distribution and random motility. These specificity studies therefore delineate three distinct and none-overlapping functions for RasG in vegetative cells.

  17. Biogenesis of lysosomal enzymes in the alpha-glucosidase II-deficient modA mutant of Dictyostelium discoideum: retention of alpha-1,3-linked glucose on N-linked oligosaccharides delays intracellular transport but does not alter sorting of alpha-mannosidase or beta-glucosidase.

    PubMed

    Ebert, D L; Bush, J M; Dimond, R L; Cardelli, J A

    1989-09-01

    The endoplasmic reticulum-localized enzyme alpha-glucosidase II is responsible for removing the two alpha-1,3-linked glucose residues from N-linked oligosaccharides of glycoproteins. This activity is missing in the modA mutant strain, M31, of Dictyostelium discoideum. Results from both radiolabeled pulse-chase and subcellular fractionation experiments indicate that this deficiency did not prevent intracellular transport and proteolytic processing of the lysosomal enzymes, alpha-mannosidase and beta-glucosidase. However, the rate at which the glucosylated precursors left the rough endoplasmic reticulum was several-fold slower than the rate at which the wild-type precursors left this compartment. Retention of glucose residues did not disrupt the binding of the precursor forms of the enzymes with intracellular membranes, indicating that the delay in movement of proteins from the ER did not result from lack of association with membranes. However, the mutant alpha-mannosidase precursor contained more trypsin-sensitive sites than did the wild-type precursor, suggesting that improper folding of precursor molecules might account for the slow rate of transport to the Golgi complex. Percoll density gradient fractionation of extracts prepared from M31 cells indicated that the proteolytically processed mature forms of alpha-mannosidase and beta-glucosidase were localized to lysosomes. Finally, the mutation in M31 may have other, more dramatic, effects on the lysosomal system since two enzymes, N-acetylglucosaminidase and acid phosphatase, were secreted much less efficiently from lysosomal compartments by the mutant strain.

  18. c-di-GMP induction of Dictyostelium cell death requires the polyketide DIF-1.

    PubMed

    Song, Yu; Luciani, Marie-Françoise; Giusti, Corinne; Golstein, Pierre

    2015-02-15

    Cell death in the model organism Dictyostelium, as studied in monolayers in vitro, can be induced by the polyketide DIF-1 or by the cyclical dinucleotide c-di-GMP. c-di-GMP, a universal bacterial second messenger, can trigger innate immunity in bacterially infected animal cells and is involved in developmental cell death in Dictyostelium. We show here that c-di-GMP was not sufficient to induce cell death in Dictyostelium cell monolayers. Unexpectedly, it also required the DIF-1 polyketide. The latter could be exogenous, as revealed by a telling synergy between c-di-GMP and DIF-1. The required DIF-1 polyketide could also be endogenous, as shown by the inability of c-di-GMP to induce cell death in Dictyostelium HMX44A cells and DH1 cells upon pharmacological or genetic inhibition of DIF-1 biosynthesis. In these cases, c-di-GMP-induced cell death was rescued by complementation with exogenous DIF-1. Taken together, these results demonstrated that c-di-GMP could trigger cell death in Dictyostelium only in the presence of the DIF-1 polyketide or its metabolites. This identified another element of control to this cell death and perhaps also to c-di-GMP effects in other situations and organisms.

  19. TipC and the chorea-acanthocytosis protein VPS13A regulate autophagy in Dictyostelium and human HeLa cells

    PubMed Central

    Muñoz-Braceras, Sandra; Calvo, Rosa; Escalante, Ricardo

    2015-01-01

    Deficient autophagy causes a distinct phenotype in Dictyostelium discoideum, characterized by the formation of multitips at the mound stage. This led us to analyze autophagy in a number of multitipped mutants described previously (tipA−, tipB−, tipC−, and tipD−). We found a clear autophagic dysfunction in tipC− and tipD− while the others showed no defects. tipD codes for a homolog of Atg16, which confirms the role of this protein in Dictyostelium autophagy and validates our approach. The tipC-encoded protein is highly similar to human VPS13A (also known as chorein), whose mutations cause the chorea-acanthocytosis syndrome. No member of the VPS13 protein family has been previously related to autophagy despite the presence of a region of similarity to Atg2 at the C terminus. This region also contains the conserved domain of unknown function DUF1162. Of interest, the expression of the TipC C-terminal coding sequence containing these 2 motifs largely complemented the mutant phenotype. Dictyostelium cells lacking TipC displayed a reduced number of autophagosomes visualized with the markers GFP-Atg18 and GFP-Atg8 and an impaired autophagic degradation as determined by a proteolytic cleavage assay. Downregulation of human VPS13A in HeLa cells by RNA interference confirmed the participation of the human protein in autophagy. VPS13A-depleted cells showed accumulation of autophagic markers and impaired autophagic flux. PMID:25996471

  20. Fucosebeta-1-P-Ser is a new type of glycosylation: using antibodies to identify a novel structure in Dictyostelium discoideum and study multiple types of fucosylation during growth and development.

    PubMed

    Srikrishna, G; Wang, L; Freeze, H H

    1998-08-01

    Three antibodies that recognize distinct fucose epitopes were used to study fucosylation during growth and development of Dictyostelium discoideum. mAb83.5 is known to recognize an undefined "fucose epitope" on several proteins with serine-rich domains, while mAb CAB4, and a component of anti-horse-radish peroxidase, specifically recognize Fucalpha1,6GlcNAc and Fucalpha1,3GlcNAc residues respectively in the core of N-linked oligosaccharides. We show that mAb 83.5 defines a new type of O-glycosylation. Serine-containing peptides incubated with GDPbeta[3H]Fuc and microsomes formed two fucosylated products. A neutral product accounting for 30% of the label did not react with the antibody, while the rest of the label was incorporated into a charged product which contained all the mAb83.5 reactive material. beta-Elimination of the labeled peptide or endogenous products produced [3H]Fuc-1-P, indicating phosphodiester linkage to serine. Fucbeta-1-P and GDP-betaFuc at 100 microM blocked mAb83.5 binding to endogenous and peptide products, but their alpha-linked anomers did not. Electrospray ionization mass spectra of the neutral and anionic labeled products showed major peaks of mass units corresponding to O-Fuc-Ser peptide and O-Fuc-phospho-Ser peptide, respectively. The activity of Fuc-phosphotransferase exactly paralleled the accumulation of reactive glycans during growth and development. The expressions of N-glycan core Fucalpha1,6GlcNAc and Fucalpha1,3GlcNAc and their respective fucosyl transferase activities were also synchronous, but their developmental regulation differed from one another. Fucalpha1, 6GlcNAc was expressed maximally during growth but declined during development. In contrast core Fucalpha1,3GlcNAc epitopes were expressed almost exclusively during development. These findings provide direct evidence for a novel type of O-phosphofucosylation, demonstrate the existence of an O-fucosyl transferase, and identify two different types of core fucosylation in

  1. Mechano-chemical signaling maintains the rapid movement of Dictyostelium cells

    SciTech Connect

    Lombardi, M.L.; Knecht, D.A.; Lee, J.

    2008-05-01

    The survival of Dictyostelium cells depends on their ability to efficiently chemotax, either towards food or to form multicellular aggregates. Although the involvement of Ca{sup 2+} signaling during chemotaxis is well known, it is not clear how this regulates cell movement. Previously, fish epithelial keratocytes have been shown to display transient increases in intracellular calcium ([Ca{sup 2+}]{sub i}) that are mediated by stretch-activated calcium channels (SACs), which play a role in retraction of the cell body [J. Lee, A. Ishihara, G. Oxford, B. Johnson, and K. Jacobson, Regulation of cell movement is mediated by stretch-activated calcium channels. Nature, 1999. 400(6742): p. 382-6.]. To investigate the involvement of SACs in Dictyostelium movement we performed high resolution calcium imaging in wild-type (NC4A2) Dictyostelium cells to detect changes in [Ca{sup 2+}]{sub i}. We observed small, brief, Ca{sup 2+} transients in randomly moving wild-type cells that were dependent on both intracellular and extracellular sources of calcium. Treatment of cells with the SAC blocker gadolinium (Gd{sup 3+}) inhibited transients and decreased cell speed, consistent with the involvement of SACs in regulating Dictyostelium motility. Additional support for SAC activity was given by the increase in frequency of Ca{sup 2+} transients when Dictyostelium cells were moving on a more adhesive substratum or when they were mechanically stretched. We conclude that mechano-chemical signaling via SACs plays a major role in maintaining the rapid movement of Dictyostelium cells.

  2. Modeling oscillations and spiral waves in Dictyostelium populations.

    PubMed

    Noorbakhsh, Javad; Schwab, David J; Sgro, Allyson E; Gregor, Thomas; Mehta, Pankaj

    2015-06-01

    Unicellular organisms exhibit elaborate collective behaviors in response to environmental cues. These behaviors are controlled by complex biochemical networks within individual cells and coordinated through cell-to-cell communication. Describing these behaviors requires new mathematical models that can bridge scales-from biochemical networks within individual cells to spatially structured cellular populations. Here we present a family of "multiscale" models for the emergence of spiral waves in the social amoeba Dictyostelium discoideum. Our models exploit new experimental advances that allow for the direct measurement and manipulation of the small signaling molecule cyclic adenosine monophosphate (cAMP) used by Dictyostelium cells to coordinate behavior in cellular populations. Inspired by recent experiments, we model the Dictyostelium signaling network as an excitable system coupled to various preprocessing modules. We use this family of models to study spatially unstructured populations of "fixed" cells by constructing phase diagrams that relate the properties of population-level oscillations to parameters in the underlying biochemical network. We then briefly discuss an extension of our model that includes spatial structure and show how this naturally gives rise to spiral waves. Our models exhibit a wide range of novel phenomena. including a density-dependent frequency change, bistability, and dynamic death due to slow cAMP dynamics. Our modeling approach provides a powerful tool for bridging scales in modeling of Dictyostelium populations.

  3. Modeling oscillations and spiral waves in Dictyostelium populations

    NASA Astrophysics Data System (ADS)

    Noorbakhsh, Javad; Schwab, David J.; Sgro, Allyson E.; Gregor, Thomas; Mehta, Pankaj

    2015-06-01

    Unicellular organisms exhibit elaborate collective behaviors in response to environmental cues. These behaviors are controlled by complex biochemical networks within individual cells and coordinated through cell-to-cell communication. Describing these behaviors requires new mathematical models that can bridge scales—from biochemical networks within individual cells to spatially structured cellular populations. Here we present a family of "multiscale" models for the emergence of spiral waves in the social amoeba Dictyostelium discoideum. Our models exploit new experimental advances that allow for the direct measurement and manipulation of the small signaling molecule cyclic adenosine monophosphate (cAMP) used by Dictyostelium cells to coordinate behavior in cellular populations. Inspired by recent experiments, we model the Dictyostelium signaling network as an excitable system coupled to various preprocessing modules. We use this family of models to study spatially unstructured populations of "fixed" cells by constructing phase diagrams that relate the properties of population-level oscillations to parameters in the underlying biochemical network. We then briefly discuss an extension of our model that includes spatial structure and show how this naturally gives rise to spiral waves. Our models exhibit a wide range of novel phenomena. including a density-dependent frequency change, bistability, and dynamic death due to slow cAMP dynamics. Our modeling approach provides a powerful tool for bridging scales in modeling of Dictyostelium populations.

  4. Lipid droplet dynamics at early stages of Mycobacterium marinum infection in Dictyostelium.

    PubMed

    Barisch, Caroline; Paschke, Peggy; Hagedorn, Monica; Maniak, Markus; Soldati, Thierry

    2015-09-01

    Lipid droplets exist in virtually every cell type, ranging not only from mammals to plants, but also to eukaryotic and prokaryotic unicellular organisms such as Dictyostelium and bacteria. They serve among other roles as energy reservoir that cells consume in times of starvation. Mycobacteria and some other intracellular pathogens hijack these organelles as a nutrient source and to build up their own lipid inclusions. The mechanisms by which host lipid droplets are captured by the pathogenic bacteria are extremely poorly understood. Using the powerful Dictyostelium discoideum/Mycobacterium marinum infection model, we observed that, immediately after their uptake, lipid droplets translocate to the vicinity of the vacuole containing live but not dead mycobacteria. Induction of lipid droplets in Dictyostelium prior to infection resulted in a vast accumulation of neutral lipids and sterols inside the bacterium-containing compartment. Subsequently, under these conditions, mycobacteria accumulated much larger lipid inclusions. Strikingly, the Dictyostelium homologue of perilipin and the murine perilipin 2 surrounded bacteria that had escaped to the cytosol of Dictyostelium or microglial BV-2 cells respectively. Moreover, bacterial growth was inhibited in Dictyostelium plnA knockout cells. In summary, our results provide evidence that mycobacteria actively manipulate the lipid metabolism of the host from very early infection stages.

  5. Intracellular killing of bacteria: is Dictyostelium a model macrophage or an alien?

    PubMed

    Cosson, Pierre; Lima, Wanessa C

    2014-06-01

    Predation of bacteria by phagocytic cells was first developed during evolution by environmental amoebae. Many of the core mechanisms used by amoebae to sense, ingest and kill bacteria have also been conserved in specialized phagocytic cells in mammalian organisms. Here we focus on recent results revealing how Dictyostelium discoideum senses and kills non-pathogenic bacteria. In this model, genetic analysis of intracellular killing of bacteria has revealed a surprisingly complex array of specialized mechanisms. These results raise new questions on these processes, and challenge current models based largely on studies in mammalian phagocytes. In addition, recent studies suggest one additional level on complexity by revealing how Dictyostelium recognizes specifically various bacterial species and strains, and adapts its metabolism to process them. It remains to be seen to what extent mechanisms uncovered in Dictyostelium are also used in mammalian phagocytic cells.

  6. Intracellular killing of bacteria: is Dictyostelium a model macrophage or an alien?

    PubMed Central

    Cosson, Pierre; Lima, Wanessa C

    2014-01-01

    Predation of bacteria by phagocytic cells was first developed during evolution by environmental amoebae. Many of the core mechanisms used by amoebae to sense, ingest and kill bacteria have also been conserved in specialized phagocytic cells in mammalian organisms. Here we focus on recent results revealing how Dictyostelium discoideum senses and kills non-pathogenic bacteria. In this model, genetic analysis of intracellular killing of bacteria has revealed a surprisingly complex array of specialized mechanisms. These results raise new questions on these processes, and challenge current models based largely on studies in mammalian phagocytes. In addition, recent studies suggest one additional level on complexity by revealing how Dictyostelium recognizes specifically various bacterial species and strains, and adapts its metabolism to process them. It remains to be seen to what extent mechanisms uncovered in Dictyostelium are also used in mammalian phagocytic cells. PMID:24628900

  7. Signal relay during the life cycle of Dictyostelium.

    PubMed

    Mahadeo, Dana C; Parent, Carole A

    2006-01-01

    A fundamental property of multicellular organisms is signal relay, the process by which information is transmitted from one cell to another. The integration of external information, such as nutritional status or developmental cues, is critical to the function of organisms. In addition, the spatial organizations of multicellular organisms require intricate signal relay mechanisms. Signal relay is remarkably exhibited during the life cycle of the social amoebae Dictyostelium discoideum, a eukaryote that retains a simple way of life, yet it has greatly contributed to our knowledge of the mechanisms cells use to communicate and integrate information. This chapter focuses on the molecules and mechanisms that Dictyostelium employs during its life cycle to relay temporal and spatial cues that are required for survival.

  8. 4D Traction Force Microscopy Reveals Asymmetric Cortical Forces in Migrating Dictyostelium Cells

    NASA Astrophysics Data System (ADS)

    Delanoë-Ayari, H.; Rieu, J. P.; Sano, M.

    2010-12-01

    We present a 4D (x; y; z; t) force map of Dictyostelium cells crawling on a soft gel substrate. Vertical forces are of the same order as the tangential ones. The cells pull the substratum upward along the cell, medium, or substratum contact line and push it downward under the cell except for the pseudopods. We demonstrate quantitatively that the variations in the asymmetry in cortical forces correlates with the variations of the direction and speed of cell displacement.

  9. Generation and characterization of Dictyostelium cells deficient in a myosin I heavy chain isoform

    PubMed Central

    1990-01-01

    Motile activities such as chemotaxis and phagocytosis, which occur in Dictyostelium cells lacking myosin II, may be dependent upon myosin I. To begin to explore this possibility, we have engineered a disruption of the Dictyostelium myosin I heavy chain (DMIHC) gene described recently (Jung, G., C. L. Saxe III, A. R. Kimmel, and J. A. Hammer III. 1989. Proc. Natl. Acad. Sci. USA. 86:6186-6190). The double-crossover, gene disruption event that occurred resulted in replacement of the middle approximate one-third of the gene with the neomycin resistance marker. The resulting cells are devoid of both the 3.6-kb DMIHC gene transcript and the 124-kD DMIHC polypeptide. DMIHC- cells are capable of chemotactic streaming and aggregation, but these processes are delayed. Furthermore, the rate of phagocytosis by DMIHC- cells is reduced, as assessed by growth rate on lawns of heat-killed bacteria and on the initial rate of uptake of FITC-labeled bacteria. Therefore, this Dictyostelium myosin I isoform appears to play a role in supporting chemotaxis and phagocytosis, but it is clearly not required for these processes to occur. Using a portion of the DMIHC gene as a probe, we have cloned three additional Dictyostelium small myosin heavy chain genes. Comparison of these four genes with three genes described recently by Titus et al. (Titus, M. A., H. M. Warrick, and J. A. Spudich. 1989. Cell Reg. 1:55-63) indicates that there are at least five small myosin heavy chain genes in Dictyostelium. The probability that there is considerable overlap of function between these small myosin isoforms indicates that multiple gene disruptions within a single cell may be necessary to generate a more striking myosin I- phenotype. PMID:2141028

  10. External stimulation strength controls actin response dynamics in Dictyostelium cells

    NASA Astrophysics Data System (ADS)

    Hsu, Hsin-Fang; Westendorf, Christian; Tarantola, Marco; Zykov, Vladimir; Bodenschatz, Eberhard; Beta, Carsten

    2015-03-01

    Self-sustained oscillation and the resonance frequency of the cytoskeletal actin polymerization/depolymerization have recently been observed in Dictyostelium, a model system for studying chemotaxis. Here we report that the resonance frequency is not constant but rather varies with the strength of external stimuli. To understand the underlying mechanism, we analyzed the polymerization and depolymerization time at different levels of external stimulation. We found that polymerization time is independent of external stimuli but the depolymerization time is prolonged as the stimulation increases. These observations can be successfully reproduced in the frame work of our time delayed differential equation model.

  11. A Cytohesin Homolog in Dictyostelium Amoebae

    PubMed Central

    Shina, Maria Christina; Müller, Rolf; Blau-Wasser, Rosemarie; Glöckner, Gernot; Schleicher, Michael; Eichinger, Ludwig; Noegel, Angelika A.; Kolanus, Waldemar

    2010-01-01

    Background Dictyostelium, an amoeboid motile cell, harbors several paralogous Sec7 genes that encode members of three distinct subfamilies of the Sec7 superfamily of Guanine nucleotide exchange factors. Among them are proteins of the GBF/BIG family present in all eukaryotes. The third subfamily represented with three members in D. discoideum is the cytohesin family that has been thought to be metazoan specific. Cytohesins are characterized by a Sec7 PH tandem domain and have roles in cell adhesion and migration. Principal Findings Dictyostelium SecG exhibits highest homologies to the cytohesins. It harbors at its amino terminus several ankyrin repeats that are followed by the Sec7 PH tandem domain. Mutants lacking SecG show reduced cell-substratum adhesion whereas cell-cell adhesion that is important for development is not affected. Accordingly, multicellular development proceeds normally in the mutant. During chemotaxis secG− cells elongate and migrate in a directed fashion towards cAMP, however speed is moderately reduced. Significance The data indicate that SecG is a relevant factor for cell-substrate adhesion and reveal the basic function of a cytohesin in a lower eukaryote. PMID:20186335

  12. How social evolution theory impacts our understanding of development in the social amoeba Dictyostelium.

    PubMed

    Strassmann, Joan E; Queller, David C

    2011-05-01

    Dictyostelium discoideum has been very useful for elucidating principles of development over the last 50 years, but a key attribute means there is a lot to be learned from a very different intellectual tradition: social evolution. Because Dictyostelium arrives at multicellularity by aggregation instead of through a single-cell bottleneck, the multicellular body could be made up of genetically distinct cells. If they are genetically distinct, natural selection will result in conflict over which cells become fertile spores and which become dead stalk cells. Evidence for this conflict includes unequal representation of two genetically different clones in spores of a chimera, the poison-like differentiation inducing factor (DIF) system that appears to involve some cells forcing others to become stalk, and reduced functionality in migrating chimeras. Understanding how selection operates on chimeras of genetically distinct clones is crucial for a comprehensive view of Dictyostelium multicellularity. In nature, Dictyostelium fruiting bodies are often clonal, or nearly so, meaning development will often be very cooperative. Relatedness levels tell us what benefits must be present for sociality to evolve. Therefore it is important to measure relatedness in nature, show that it has an impact on cooperation in the laboratory, and investigate genes that Dictyostelium uses to discriminate between relatives and non-relatives. Clearly, there is a promising future for research at the interface of development and social evolution in this fascinating group.

  13. Identification of sequences regulating the transcription of a Dictyostelium gene selectively expressed in prespore cells.

    PubMed Central

    Early, A E; Williams, J G

    1989-01-01

    There has been considerable debate about the relative contributions of transcriptional and post-transcriptional mechanisms to the regulation of prespore gene expression in Dictyostelium. We have determined the DNA sequence upstream of D19, the Dictyostelium gene encoding PsA, a prespore-specific, cell surface protein of unknown function. Our analysis of gene fusions, in which D19 upstream sequences are placed adjacent to a heterologous reporter gene, indicates that transcriptional signals alone are sufficient for the correct temporal and cell-type specific expression of this gene. We also show that the 5' and 3' boundaries of the minimal sequences necessary for correct developmental regulation lie within the region 338 to 122 nucleotides upstream of the start site of transcription but that flanking sequences seem to be necessary for optimal expression. Images PMID:2550894

  14. Expression of activated Ras during Dictyostelium development alters cell localization and changes cell fate.

    PubMed

    Jaffer, Z M; Khosla, M; Spiegelman, G B; Weeks, G

    2001-03-01

    There is now a body of evidence to indicate that Ras proteins play important roles in development. Dictyostelium expresses several ras genes and each appears to perform a distinct function. Previous data had indicated that the overexpression of an activated form of the major developmentally regulated gene, rasD, caused a major aberration in morphogenesis and cell type determination. We now show that the developmental expression of an activated rasG gene under the control of the rasD promoter causes a similar defect. Our results indicate that the expression of activated rasG in prespore cells results in their transdifferentiation into prestalk cells, whereas activated rasG expression in prestalk causes gross mislocalization of the prestalk cell populations.

  15. Bitter tastant responses in the amoeba Dictyostelium correlate with rat and human taste assays.

    PubMed

    Cocorocchio, Marco; Ives, Robert; Clapham, David; Andrews, Paul L R; Williams, Robin S B

    2016-01-01

    Treatment compliance is reduced when pharmaceutical compounds have a bitter taste and this is particularly marked for paediatric medications. Identification of bitter taste liability during drug discovery utilises the rat in vivo brief access taste aversion (BATA) test which apart from animal use is time consuming with limited throughput. We investigated the suitability of using a simple, non-animal model, the amoeba Dictyostelium discoideum to investigate taste-related responses and particularly identification of compounds with a bitter taste liability. The effect of taste-related compounds on Dictyostelium behaviour following acute exposure (15 minutes) was monitored. Dictyostelium did not respond to salty, sour, umami or sweet tasting compounds, however, cells rapidly responded to bitter tastants. Using time-lapse photography and computer-generated quantification to monitor changes in cell membrane movement, we developed an assay to assess the response of Dictyostelium to a wide range of structurally diverse known bitter compounds and blinded compounds. Dictyostelium showed varying responses to the bitter tastants, with IC50 values providing a rank order of potency. Comparison of Dictyostelium IC50 values to those observed in response to a similar range of compounds in the rat in vivo brief access taste aversion test showed a significant (p = 0.0172) positive correlation between the two models, and additionally a similar response to that provided by a human sensory panel assessment test. These experiments demonstrate that Dictyostelium may provide a suitable model for early prediction of bitterness for novel tastants and drugs. Interestingly, a response to bitter tastants appears conserved from single-celled amoebae to humans.

  16. Dictyostelium Lipid Droplets Host Novel Proteins

    PubMed Central

    Du, Xiaoli; Barisch, Caroline; Paschke, Peggy; Herrfurth, Cornelia; Bertinetti, Oliver; Pawolleck, Nadine; Otto, Heike; Rühling, Harald; Feussner, Ivo; Herberg, Friedrich W.

    2013-01-01

    Across all kingdoms of life, cells store energy in a specialized organelle, the lipid droplet. In general, it consists of a hydrophobic core of triglycerides and steryl esters surrounded by only one leaflet derived from the endoplasmic reticulum membrane to which a specific set of proteins is bound. We have chosen the unicellular organism Dictyostelium discoideum to establish kinetics of lipid droplet formation and degradation and to further identify the lipid constituents and proteins of lipid droplets. Here, we show that the lipid composition is similar to what is found in mammalian lipid droplets. In addition, phospholipids preferentially consist of mainly saturated fatty acids, whereas neutral lipids are enriched in unsaturated fatty acids. Among the novel protein components are LdpA, a protein specific to Dictyostelium, and Net4, which has strong homologies to mammalian DUF829/Tmem53/NET4 that was previously only known as a constituent of the mammalian nuclear envelope. The proteins analyzed so far appear to move from the endoplasmic reticulum to the lipid droplets, supporting the concept that lipid droplets are formed on this membrane. PMID:24036346

  17. Adenylyl cyclase localization to the uropod of aggregating Dictyostelium cells requires RacC

    PubMed Central

    Wang, C.; Jung, D.; Cao, Z.; Chung, C. Y.

    2015-01-01

    The localization of adenylyl cyclase A (ACA) to uropod of cells is required for the stream formation during Dictyostelium development. RacC is a Dictyostelium orthologue of Cdc42. We identified a streaming defect of racC− cells as they are clearly less polarized and form smaller and fragmented streams. ACA-YFP is mainly associated with intracellular vesicular structures, but not with the plasma membrane in racC− cells. racC− cells have a slightly higher number of vesicles than Ax3 cells, suggesting that the defect of ACA trafficking is not simply due to the lack of vesicle formation. While the ACA-YFP vesicles traveled with an average velocity of 9.1 µm/min in Ax3 cells, a slow and diffusional movement without direction with an average velocity of 4 µm/min was maintained in racC− cells. Images acquired by using total internal reflection fluorescence (TIRF) microscopy and fluorescence recovery after photobleaching (FRAP) analysis revealed that a significantly decreased number of ACA-YFP vesicles appeared near the cell membrane, indicating a defect in ACA-YFP vesicle trafficking. These results suggest an important role of RacC in the rapid and directional movements of ACA vesicles on microtubules to the plasma membrane, especially to the back of polarized cell. PMID:26315268

  18. Adenylyl cyclase localization to the uropod of aggregating Dictyostelium cells requires RacC.

    PubMed

    Wang, C; Jung, D; Cao, Z; Chung, C Y

    2015-09-25

    The localization of adenylyl cyclase A (ACA) to uropod of cells is required for the stream formation during Dictyostelium development. RacC is a Dictyostelium orthologue of Cdc42. We identified a streaming defect of racC(-) cells as they are clearly less polarized and form smaller and fragmented streams. ACA-YFP is mainly associated with intracellular vesicular structures, but not with the plasma membrane in racC(-) cells. racC(-) cells have a slightly higher number of vesicles than Ax3 cells, suggesting that the defect of ACA trafficking is not simply due to the lack of vesicle formation. While the ACA-YFP vesicles traveled with an average velocity of 9.1 μm/min in Ax3 cells, a slow and diffusional movement without direction with an average velocity of 4 μm/min was maintained in racC(-) cells. Images acquired by using total internal reflection fluorescence (TIRF) microscopy and fluorescence recovery after photobleaching (FRAP) analysis revealed that a significantly decreased number of ACA-YFP vesicles appeared near the cell membrane, indicating a defect in ACA-YFP vesicle trafficking. These results suggest an important role of RacC in the rapid and directional movements of ACA vesicles on microtubules to the plasma membrane, especially to the back of polarized cell.

  19. Identification of regions essential for extrachromosomal replication and maintenance of an endogenous plasmid in Dictyostelium.

    PubMed Central

    Ahern, K G; Howard, P K; Firtel, R A

    1988-01-01

    Initial experiments with the endogenous 12.3 kb Dictyostelium discoideum plasmid Ddp1 led to the generation of a large shuttle vector, Ddp1-20. In addition to Ddp1, this vector contains pBR322 and a gene fusion that confers G418 resistance in Dictyostelium cells. We have shown that Ddp1-20 replicates extrachromosomally in Dictyostelium cells and can be grown in Escherichia coli cells (1). We have now examined deletions within this vector to identify the elements essential for extrachromosomal replication and stable maintenance of the plasmid. We find that a 2.2 kb fragment is sufficient to confer stable, extrachromosomal replication with a reduction in copy number from about 40 to approximately 10-15 copies per cell. Vectors containing additional Ddp1 sequences have a higher copy number. The 2.2 kb region contains none of the complete, previously identified transcription units on Ddp1 expressed during vegetative growth or development. These results suggest that gene products expressed by Ddp1 are not essential for replication, stability, or partitioning of the plasmid between daughter cells. Vectors carrying only the 2.2 kb fragment plus the gene fusion conferring G418 resistance transform Dictyostelium cells with high efficiency using either calcium phosphate mediated transformation or electroporation. Finally, we have examined the relative levels of expression of actin promoters driving neoR genes when in extrachromosomal or integrating vectors. Images PMID:3405751

  20. Gdt2 regulates the transition of Dictyostelium cells from growth to differentiation

    PubMed Central

    Chibalina, Margarita V; Anjard, Christophe; Insall, Robert H

    2004-01-01

    Background Dictyostelium life cycle consists of two distinct phases – growth and development. The control of growth-differentiation transition in Dictyostelium is not completely understood, and only few genes involved in this process are known. Results We have isolated a REMI (restriction enzyme-mediated integration) mutant, which prematurely initiates multicellular development. When grown on a bacterial lawn, these cells aggregate before the bacteria are completely cleared. In bacterial suspension, mutant cells express the developmental marker discoidin Iγ even at low cell densities and high concentrations of bacteria. In the absence of nutrients, mutant cells aggregate more rapidly than wild type, but the rest of development is unaffected and normal fruiting bodies are formed. The disrupted gene shows substantial homology to the recently described gdt1 gene, and therefore was named gdt2. While GDT1 and GDT2 are similar in many ways, there are intriguing differences. GDT2 contains a well conserved protein kinase domain, unlike GDT1, whose kinase domain is probably non-functional. The gdt2 and gdt1 mRNAs are regulated differently, with gdt2 but not gdt1 expressed throughout development. The phenotypes of gdt2- and gdt1- mutants are related but not identical. While both initiate development early, gdt2- cells grow at a normal rate, unlike gdt1- mutants. Protein kinase A levels and activity are essentially normal in growing gdt2- mutants, implying that GDT2 regulates a pathway that acts separately from PKA. Gdt1 and gdt2 are the first identified members of a family containing at least eight closely related genes. Conclusions We have isolated and characterised a new gene, gdt2, which acts to restrain development until conditions are appropriate. We also described a family of related genes in the Dictyostelium genome. We hypothesise that different family members might control similar cellular processes, but respond to different environmental cues. PMID:15236669

  1. Cell-substrate interactions and locomotion of Dictyostelium wild-type and mutants defective in three cytoskeletal proteins: a study using quantitative reflection interference contrast microscopy.

    PubMed Central

    Schindl, M; Wallraff, E; Deubzer, B; Witke, W; Gerisch, G; Sackmann, E

    1995-01-01

    Reflection interference contrast microscopy combined with digital image processing was applied to study the motion of Dictyostelium discoideum cells in their pre-aggregative state on substrata of different adhesiveness (glass, albumin-covered glass, and freshly cleaved mica). The temporal variations of the size and shape of the cell/substratum contact area and the time course of advancement of pseudopods protruding in contact with the substratum were analyzed. The major goal was to study differences between the locomotion of wild-type cells and strains of triple mutants deficient in two F-actin cross-linking proteins (alpha-actinin and the 120-kDa gelation factor) and one F-actin fragmenting protein (severin). The size of contact area, AC, of both wild-type and mutant cells fluctuates between minimum and maximum values on the order of minutes, pointing toward an intrinsic switching mechanism associated with the mechanochemical control system. The fluctuation amplitudes are much larger on freshly cleaved mica than on glass. Wild-type and mutant cells exhibit remarkable differences on mica but not on glass. These differences comprise the population median of AC and alterations in pseudopod protrusion. AC is smaller by a factor of two or more for all mutants. Pseudopods protrude slower and shorter in the mutants. It is concluded that cell shape and pseudopods are destabilized by defects in the actin-skeleton, which can be overcompensated by strongly adhesive substrata. Several features of amoeboid cell locomotion on substrata can be understood on the basis of the minimum bending energy concept of soft adhering shells and by assuming that adhesion induces local alterations of the composite membrane consisting of the protein/lipid bilayer on the cell surface and the underlying actin-cortex. Images FIGURE 3 FIGURE 4 FIGURE 6 FIGURE 8 FIGURE 9 FIGURE 10 FIGURE 11 FIGURE 12 PMID:7756537

  2. Dissecting the function of Atg1 complex in Dictyostelium autophagy reveals a connection with the pentose phosphate pathway enzyme transketolase

    PubMed Central

    Mesquita, Ana; Tábara, Luis C.; Martinez-Costa, Oscar; Santos-Rodrigo, Natalia; Vincent, Olivier; Escalante, Ricardo

    2015-01-01

    The network of protein–protein interactions of the Dictyostelium discoideum autophagy pathway was investigated by yeast two-hybrid screening of the conserved autophagic proteins Atg1 and Atg8. These analyses confirmed expected interactions described in other organisms and also identified novel interactors that highlight the complexity of autophagy regulation. The Atg1 kinase complex, an essential regulator of autophagy, was investigated in detail here. The composition of the Atg1 complex in D. discoideum is more similar to mammalian cells than to Saccharomyces cerevisiae as, besides Atg13, it contains Atg101, a protein not conserved in this yeast. We found that Atg101 interacts with Atg13 and genetic disruption of these proteins in Dictyostelium leads to an early block in autophagy, although the severity of the developmental phenotype and the degree of autophagic block is higher in Atg13-deficient cells. We have also identified a protein containing zinc-finger B-box and FNIP motifs that interacts with Atg101. Disruption of this protein increases autophagic flux, suggesting that it functions as a negative regulator of Atg101. We also describe the interaction of Atg1 kinase with the pentose phosphate pathway enzyme transketolase (TKT). We found changes in the activity of endogenous TKT activity in strains lacking or overexpressing Atg1, suggesting the presence of an unsuspected regulatory pathway between autophagy and the pentose phosphate pathway in Dictyostelium that seems to be conserved in mammalian cells. PMID:26246495

  3. Dissecting the function of Atg1 complex in Dictyostelium autophagy reveals a connection with the pentose phosphate pathway enzyme transketolase.

    PubMed

    Mesquita, Ana; Tábara, Luis C; Martinez-Costa, Oscar; Santos-Rodrigo, Natalia; Vincent, Olivier; Escalante, Ricardo

    2015-08-01

    The network of protein-protein interactions of the Dictyostelium discoideum autophagy pathway was investigated by yeast two-hybrid screening of the conserved autophagic proteins Atg1 and Atg8. These analyses confirmed expected interactions described in other organisms and also identified novel interactors that highlight the complexity of autophagy regulation. The Atg1 kinase complex, an essential regulator of autophagy, was investigated in detail here. The composition of the Atg1 complex in D. discoideum is more similar to mammalian cells than to Saccharomyces cerevisiae as, besides Atg13, it contains Atg101, a protein not conserved in this yeast. We found that Atg101 interacts with Atg13 and genetic disruption of these proteins in Dictyostelium leads to an early block in autophagy, although the severity of the developmental phenotype and the degree of autophagic block is higher in Atg13-deficient cells. We have also identified a protein containing zinc-finger B-box and FNIP motifs that interacts with Atg101. Disruption of this protein increases autophagic flux, suggesting that it functions as a negative regulator of Atg101. We also describe the interaction of Atg1 kinase with the pentose phosphate pathway enzyme transketolase (TKT). We found changes in the activity of endogenous TKT activity in strains lacking or overexpressing Atg1, suggesting the presence of an unsuspected regulatory pathway between autophagy and the pentose phosphate pathway in Dictyostelium that seems to be conserved in mammalian cells.

  4. IQGAP Family Members in Yeast, Dictyostelium, and Mammalian Cells

    PubMed Central

    Shannon, Katie B.

    2012-01-01

    IQGAPs are a family of scaffolding proteins with multiple domains, named for the IQ motifs and GTPase activating protein (GAP) related domains. Despite their GAP homology, IQGAP proteins act as effectors for GTP-bound GTPases of the Ras superfamily and do not stimulate GTP hydrolysis. IQGAPs are found in eukaryotic cells from yeast to human, and localize to actin-containing structures such as lamellipodia, membrane ruffles, cell-cell adhesions, phagocytic cups, and the actomyosin ring formed during cytokinesis. Mammalian IQGAPs also act as scaffolds for signaling pathways. IQGAPs perform their myriad functions through association with a large number of proteins including filamentous actin (F-actin), GTPases, calcium-binding proteins, microtubule binding proteins, kinases, and receptors. The focus of this paper is on recent studies describing new binding partners, mechanisms of regulation, and biochemical and physiological functions of IQGAPs in yeast, amoeba, and mammalian cells. PMID:22505937

  5. Robustness of self-organizing chemoattractant field arising from precise pulse induction of its breakdown enzyme: a single-cell level analysis of PDE expression in Dictyostelium.

    PubMed

    Masaki, Noritaka; Fujimoto, Koichi; Honda-Kitahara, Mai; Hada, Emi; Sawai, Satoshi

    2013-03-05

    The oscillation of chemoattractant cyclic AMP (cAMP) in Dictyostelium discoideum is a collective phenomenon that occurs when the basal level of extracellular cAMP exceeds a threshold and invokes cooperative mutual excitation of cAMP synthesis and secretion. For pulses to be relayed from cell to cell repetitively, secreted cAMP must be cleared and brought down to the subthreshold level. One of the main determinants of the oscillatory behavior is thus how much extracellular cAMP is degraded by extracellular phosphodiesterase (PDE). To date, the exact nature of PDE gene regulation remains elusive. Here, we performed live imaging analysis of mRNA transcripts for pdsA--the gene encoding extracellular PDE. Our analysis revealed that pdsA is upregulated during the rising phase of cAMP oscillations. Furthermore, by analyzing isolated cells, we show that expression of pdsA is strictly dependent on the presence of extracellular cAMP. pdsA is induced only at ∼1 nM extracellular cAMP, which is almost identical to the threshold concentration for the cAMP relay response. The observed precise regulation of PDE expression together with degradation of extracellular cAMP by PDE form a dual positive and negative feedback circuit, and model analysis shows that this sets the cAMP level near the threshold concentration for the cAMP relay response for a wide range of adenylyl cyclase activity. The overlap of the thresholds could allow oscillations of chemoattractant cAMP to self-organize at various starving conditions, making its development robust to fluctuations in its environment.

  6. Role of PKD2 in rheotaxis in Dictyostelium.

    PubMed

    Lima, Wanessa C; Vinet, Adrien; Pieters, Jean; Cosson, Pierre

    2014-01-01

    The sensing of mechanical forces modulates several cellular responses as adhesion, migration and differentiation. Transient elevations of calcium concentration play a key role in the activation of cells following mechanical stress, but it is still unclear how eukaryotic cells convert a mechanical signal into an ion flux. In this study, we used the model organism Dictyostelium discoideum to assess systematically the role of individual calcium channels in mechanosensing. Our results indicate that PKD2 is the major player in the cell response to rheotaxis (i.e., shear-flow induced mechanical motility), while other putative calcium channels play at most minor roles. Mutant pkd2 KO cells lose the ability to orient relative to a shear flow, whereas their ability to move towards a chemoattractant is unaffected. PKD2 is also important for calcium-induced lysosome exocytosis: WT cells show a transient, 2-fold increase in lysosome secretion upon sudden exposure to high levels of extracellular calcium, but pkd2 KO cells do not. In Dictyostelium, PKD2 is specifically localized at the plasma membrane, where it may generate calcium influxes in response to mechanical stress or extracellular calcium changes.

  7. Transmembrane myosin chitin synthase involved in mollusc shell formation produced in Dictyostelium is active

    SciTech Connect

    Schoenitzer, Veronika; Eichner, Norbert; Clausen-Schaumann, Hauke; Weiss, Ingrid M.

    2011-12-02

    Highlights: Black-Right-Pointing-Pointer Dictyostelium produces the 264 kDa myosin chitin synthase of bivalve mollusc Atrina. Black-Right-Pointing-Pointer Chitin synthase activity releases chitin, partly associated with the cell surface. Black-Right-Pointing-Pointer Membrane extracts of transgenic slime molds produce radiolabeled chitin in vitro. Black-Right-Pointing-Pointer Chitin producing Dictyostelium cells can be characterized by atomic force microscopy. Black-Right-Pointing-Pointer This model system enables us to study initial processes of chitin biomineralization. -- Abstract: Several mollusc shells contain chitin, which is formed by a transmembrane myosin motor enzyme. This protein could be involved in sensing mechanical and structural changes of the forming, mineralizing extracellular matrix. Here we report the heterologous expression of the transmembrane myosin chitin synthase Ar-CS1 of the bivalve mollusc Atrina rigida (2286 amino acid residues, M.W. 264 kDa/monomer) in Dictyostelium discoideum, a model organism for myosin motor proteins. Confocal laser scanning immunofluorescence microscopy (CLSM), chitin binding GFP detection of chitin on cells and released to the cell culture medium, and a radiochemical activity assay of membrane extracts revealed expression and enzymatic activity of the mollusc chitin synthase in transgenic slime mold cells. First high-resolution atomic force microscopy (AFM) images of Ar-CS1 transformed cellulose synthase deficient D. discoideumdcsA{sup -} cell lines are shown.

  8. Actin binding domain of filamin distinguishes posterior from anterior actin filaments in migrating Dictyostelium cells

    PubMed Central

    Shibata, Keitaro; Nagasaki, Akira; Adachi, Hiroyuki; Uyeda, Taro Q. P.

    2016-01-01

    Actin filaments in different parts of a cell interact with specific actin binding proteins (ABPs) and perform different functions in a spatially regulated manner. However, the mechanisms of those spatially-defined interactions have not been fully elucidated. If the structures of actin filaments differ in different parts of a cell, as suggested by previous in vitro structural studies, ABPs may distinguish these structural differences and interact with specific actin filaments in the cell. To test this hypothesis, we followed the translocation of the actin binding domain of filamin (ABDFLN) fused with photoswitchable fluorescent protein (mKikGR) in polarized Dictyostelium cells. When ABDFLN-mKikGR was photoswitched in the middle of a polarized cell, photoswitched ABDFLN-mKikGR rapidly translocated to the rear of the cell, even though actin filaments were abundant in the front. The speed of translocation (>3 μm/s) was much faster than that of the retrograde flow of cortical actin filaments. Rapid translocation of ABDFLN-mKikGR to the rear occurred normally in cells lacking GAPA, the only protein, other than actin, known to bind ABDFLN. We suggest that ABDFLN recognizes a certain feature of actin filaments in the rear of the cell and selectively binds to them, contributing to the posterior localization of filamin.

  9. Two distinct sensing pathways allow recognition of Klebsiella pneumoniae by Dictyostelium amoebae.

    PubMed

    Lima, Wanessa C; Balestrino, Damien; Forestier, Christiane; Cosson, Pierre

    2014-03-01

    Recognition of bacteria by metazoans is mediated by receptors that recognize different types of microorganisms and elicit specific cellular responses. The soil amoebae Dictyostelium discoideum feeds upon a variable mixture of environmental bacteria, and it is expected to recognize and adapt to various food sources. To date, however, no bacteria-sensing mechanisms have been described. In this study, we isolated a Dictyostelium mutant (fspA KO) unable to grow in the presence of non-capsulated Klebsiella pneumoniae bacteria, but growing as efficiently as wild-type cells in the presence of other bacteria, such as Bacillus subtilis. fspA KO cells were also unable to respond to K. pneumoniae and more specifically to bacterially secreted folate in a chemokinetic assay, while they responded readily to B. subtilis. Remarkably, both WT and fspA KO cells were able to grow in the presence of capsulated LM21 K. pneumoniae, and responded to purified capsule, indicating that capsule recognition may represent an alternative, FspA-independent mechanism for K. pneumoniae sensing. When LM21 capsule synthesis genes were deleted, growth and chemokinetic response were lost for fspA KO cells, but not for WT cells. Altogether, these results indicate that Dictyostelium amoebae use specific recognition mechanisms to respond to different K. pneumoniae elements.

  10. Conserved valproic-acid-induced lipid droplet formation in Dictyostelium and human hepatocytes identifies structurally active compounds.

    PubMed

    Elphick, Lucy M; Pawolleck, Nadine; Guschina, Irina A; Chaieb, Leila; Eikel, Daniel; Nau, Heinz; Harwood, John L; Plant, Nick J; Williams, Robin S B

    2012-03-01

    Lipid droplet formation and subsequent steatosis (the abnormal retention of lipids within a cell) has been reported to contribute to hepatotoxicity and is an adverse effect of many pharmacological agents including the antiepileptic drug valproic acid (VPA). In this study, we have developed a simple model system (Dictyostelium discoideum) to investigate the effects of VPA and related compounds in lipid droplet formation. In mammalian hepatocytes, VPA increases lipid droplet accumulation over a 24-hour period, giving rise to liver cell damage, and we show a similar effect in Dictyostelium following 30 minutes of VPA treatment. Using (3)H-labelled polyunsaturated (arachidonic) or saturated (palmitic) fatty acids, we shown that VPA treatment of Dictyostelium gives rise to an increased accumulation of both types of fatty acids in phosphatidylcholine, phosphatidylethanolamine and non-polar lipids in this time period, with a similar trend observed in human hepatocytes (Huh7 cells) labelled with [(3)H]arachidonic acid. In addition, pharmacological inhibition of β-oxidation in Dictyostelium phenocopies fatty acid accumulation, in agreement with data reported in mammalian systems. Using Dictyostelium, we then screened a range of VPA-related compounds to identify those with high and low lipid-accumulation potential, and validated these activities for effects on lipid droplet formation by using human hepatocytes. Structure-activity relationships for these VPA-related compounds suggest that lipid accumulation is independent of VPA-catalysed teratogenicity and inositol depletion. These results suggest that Dictyostelium could provide both a novel model system for the analysis of lipid droplet formation in human hepatocytes and a rapid method for identifying VPA-related compounds that show liver toxicology.

  11. Expression of an activated rasD gene changes cell fate decisions during Dictyostelium development.

    PubMed

    Louis, S A; Spiegelman, G B; Weeks, G

    1997-02-01

    It has been previously demonstrated that the expression of an activated rasD gene in wild-type Dictyostelium cells results in formation of aggregates with multitips, instead of the normal single tips, and a block in further development. In an attempt to better understand the role of activated RasD development, we examined cell-type-specific gene expression in a strain stably expressing high levels of RasD[G12T]. We found that the expression of prestalk cell-specific genes ecmA and tagB was markedly enhanced, whereas the expression of the prespore cell-specific gene cotC was reduced to very low levels. When the fate of cells in the multitipped aggregate was monitored with an ecmA/lacZ fusion, it appeared that most of the cells eventually adopted prestalk gene expression characteristics. When mixtures of the [G12T]rasD cells and Ax3 cells were induced to differentiate, chimeric pseudoplasmodia were not formed. Thus, although the [G12T]rasD transformant had a marked propensity to form prestalk cells, it could not supply the prestalk cell population when mixed with wild-type cells. Both stalk and spore cell formation occurred in low cell density monolayers of the [G12T]rasD strain, suggesting that at least part of the inhibition of stalk and spore formation during multicellular development involved inhibitory cell interactions within the cell mass. Models for the possible role of rasD in development are discussed.

  12. Developmental significance of cyanide-resistant respiration under stressed conditions: experiments in Dictyostelium cells.

    PubMed

    Kimura, Kei; Kuwayama, Hidekazu; Amagai, Aiko; Maeda, Yasuo

    2010-09-01

    We have previously reported that benzohydroxamic acid (BHAM), a potent inhibitor of cyanide (CN)-resistant respiration mediated by alternative oxidase (AOX), induces formation of unique cell masses (i.e., stalk-like cells with a large vacuole and thick cell wall) in starved Dictyostelium cells. Unexpectedly, however, aox-null cells prepared by homologous recombination exhibited normal development under normal culture conditions on agar, indicating that BHAM-induced stalk formation is not solely attributable to inhibition of CN-resistant respiration. This also suggests that a series of pharmacological approaches in the field of life science has serious limitations. Under stress (e.g., in submerged culture), starved aox-null cells exhibited slightly delayed aggregation compared with parental Ax-2 cells; most cells remained as loose aggregates even after prolonged incubation. Also, the developmental defects of aox-null cells became more marked upon incubation for 30 min just after starvation in the presence of ≥ 1.75 mmol/L H(2)O(2). This seems to indicate that CN-resistant respiration could mitigate cellular damage through reactive oxygen species (ROS), because AOX has a potential role in reduction of ROS production. Starved aox-null cells did not develop in the presence of 5 mmol/L KCN (which completely inhibited the conventional cytochrome-mediated respiration) and remained as non-aggregated single cells on agar even after prolonged incubation. Somewhat surprisingly, however, parental Ax-2 cells were found to develop normally, forming fruiting bodies even in the presence of 10 mmol/L KCN. Taken together, these results suggest that CN-resistant respiration might compensate for the production of adenosine tri-phosphate via oxidative phosphorylation.

  13. The cyclase-associated protein CAP as regulator of cell polarity and cAMP signaling in Dictyostelium.

    PubMed

    Noegel, Angelika A; Blau-Wasser, Rosemarie; Sultana, Hameeda; Müller, Rolf; Israel, Lars; Schleicher, Michael; Patel, Hitesh; Weijer, Cornelis J

    2004-02-01

    Cyclase-associated protein (CAP) is an evolutionarily conserved regulator of the G-actin/F-actin ratio and, in yeast, is involved in regulating the adenylyl cyclase activity. We show that cell polarization, F-actin organization, and phototaxis are altered in a Dictyostelium CAP knockout mutant. Furthermore, in complementation assays we determined the roles of the individual domains in signaling and regulation of the actin cytoskeleton. We studied in detail the adenylyl cyclase activity and found that the mutant cells have normal levels of the aggregation phase-specific adenylyl cyclase and that receptor-mediated activation is intact. However, cAMP relay that is responsible for the generation of propagating cAMP waves that control the chemotactic aggregation of starving Dictyostelium cells was altered, and the cAMP-induced cGMP production was significantly reduced. The data suggest an interaction of CAP with adenylyl cyclase in Dictyostelium and an influence on signaling pathways directly as well as through its function as a regulatory component of the cytoskeleton.

  14. Ras Proteins Have Multiple Functions in Vegetative Cells of Dictyostelium

    PubMed Central

    Bolourani, Parvin; Spiegelman, George; Weeks, Gerald

    2010-01-01

    During the aggregation of Dictyostelium cells, signaling through RasG is more important in regulating cyclic AMP (cAMP) chemotaxis, whereas signaling through RasC is more important in regulating the cAMP relay. However, RasC is capable of substituting for RasG for chemotaxis, since rasG− cells are only partially deficient in chemotaxis, whereas rasC−/rasG− cells are totally incapable of chemotaxis. In this study we have examined the possible functional overlap between RasG and RasC in vegetative cells by comparing the vegetative cell properties of rasG−, rasC−, and rasC−/rasG− cells. In addition, since RasD, a protein not normally found in vegetative cells, is expressed in vegetative rasG− and rasC−/rasG− cells and appears to partially compensate for the absence of RasG, we have also examined the possible functional overlap between RasG and RasD by comparing the properties of rasG− and rasC−/rasG− cells with those of the mutant cells expressing higher levels of RasD. The results of these two lines of investigation show that RasD is capable of totally substituting for RasG for cytokinesis and growth in suspension, whereas RasC is without effect. In contrast, for chemotaxis to folate, RasC is capable of partially substituting for RasG, but RasD is totally without effect. Finally, neither RasC nor RasD is able to substitute for the role that RasG plays in regulating actin distribution and random motility. These specificity studies therefore delineate three distinct and none-overlapping functions for RasG in vegetative cells. PMID:20833893

  15. The Dictyostelium Kinome—Analysis of the Protein Kinases from a Simple Model Organism

    PubMed Central

    Liu, Allen; Fey, Petra; Pilcher, Karen E; Xu, Yanji; Smith, Janet L

    2006-01-01

    Dictyostelium discoideum is a widely studied model organism with both unicellular and multicellular forms in its developmental cycle. The Dictyostelium genome encodes 285 predicted protein kinases, similar to the count of the much more advanced Drosophila. It contains members of most kinase classes shared by fungi and metazoans, as well as many previously thought to be metazoan specific, indicating that they have been secondarily lost from the fungal lineage. This includes the entire tyrosine kinase–like (TKL) group, which is expanded in Dictyostelium and includes several novel receptor kinases. Dictyostelium lacks tyrosine kinase group kinases, and most tyrosine phosphorylation appears to be mediated by TKL kinases. About half of Dictyostelium kinases occur in subfamilies not present in yeast or metazoa, suggesting that protein kinases have played key roles in the adaptation of Dictyostelium to its habitat. This study offers insights into kinase evolution and provides a focus for signaling analysis in this system. PMID:16596165

  16. The effects of expression of an activated rasG mutation on the differentiation of Dictyostelium.

    PubMed

    Thiery, R; Robbins, S; Khosla, M; Spiegelman, G B; Weeks, G

    1992-01-01

    Dictyostelium discoideum contains two ras genes, rasG and rasD, that are expressed during growth and differentiation, respectively. It was shown previously that Dictyostelium transformants expressing an activated rasD gene (a mutation producing a change in amino acid 12 from glycine to threonine) developed abnormally. When developed on filters these transformants formed multitipped aggregates, which did not go on to produce final fruiting bodies, but in a submerged culture assay on a plastic surface they either formed small aggregates or did not aggregate. In this study we transformed cells with the rasG gene, mutated to change amino acid 12 from glycine to threonine. The resulting transformants developed normally on filters, but aggregation under other conditions was impaired. In particular, in submerged culture on a plastic surface they either produced very small aggregates or did not aggregate, one of the phenotypes exhibited by the activated rasD transformants. Molecular analysis of the transformants revealed the presence of high copy numbers of the mutated rasG gene, but the level of expression of the mutant gene never exceeded the level of expression of the endogenous gene. These results indicate a powerful dominant effect of a relatively small amount of the activated RasG protein in Dictyostelium.

  17. Morphological changes and depressed phagocytic efficiency in Dictyostelium amoebae treated with toxic concentrations of cadmium

    SciTech Connect

    Cyr, R.J.; Bernstein, R.L.

    1984-10-01

    The morphology and phagocytic efficiency of Dictyostelium discoideum amoebae exposed to cadmium was investigated at two Cd concentrations: a low toxic concentration - 7 x 10/sup -5/ m, and a high toxic concentration - 2 x 10/sup -4/ m. Both concentrations inhibited growth completely; however, only in the culture containing a high toxic concentration of cadmium were severe ultrastructural anomalies observed, notably, nucleolar changes and autophagic vacuolar formation. Using biological indices it was concluded that the high concentration of cadmium was lethal and that morphological changes associated with this dose of cadmium may be secondary to cell death. In contrast, amoebae treated with a low toxic but nonlethal concentration of Cd showed an altered size distribution of cytoplasmic vacuoles and a decreased phagocytic efficiency. Cultures whose growth was completely inhibited with cobalt were also examined, as were untreated control cultures. By 24 hr Cd-treated amoebae showed a 20% decrease in the cytoplasmic mean-vacuolar diameter and a 69% decrease in phagocytic efficiency whereas Co and untreated controls showed no significant decrease in the cytoplasmic mean-vacuolar diameter. Phagocytic efficiency was only slightly diminished by Co. Changes in vacuolar profiles had been shown earlier to be related to membrane utilization in Dictyostelium amoebae. Cd at low toxic concentrations affects membrane function in Dictyostelium amoebae.

  18. Transmembrane myosin chitin synthase involved in mollusc shell formation produced in Dictyostelium is active.

    PubMed

    Schönitzer, Veronika; Eichner, Norbert; Clausen-Schaumann, Hauke; Weiss, Ingrid M

    2011-12-02

    Several mollusc shells contain chitin, which is formed by a transmembrane myosin motor enzyme. This protein could be involved in sensing mechanical and structural changes of the forming, mineralizing extracellular matrix. Here we report the heterologous expression of the transmembrane myosin chitin synthase Ar-CS1 of the bivalve mollusc Atrina rigida (2286 amino acid residues, M.W. 264 kDa/monomer) in Dictyostelium discoideum, a model organism for myosin motor proteins. Confocal laser scanning immunofluorescence microscopy (CLSM), chitin binding GFP detection of chitin on cells and released to the cell culture medium, and a radiochemical activity assay of membrane extracts revealed expression and enzymatic activity of the mollusc chitin synthase in transgenic slime mold cells. First high-resolution atomic force microscopy (AFM) images of Ar-CS1 transformed cellulose synthase deficient D. discoideumdcsA(-) cell lines are shown.

  19. Learning Physics of Living Systems from Dictyostelium

    PubMed Central

    Levine, Herbert

    2014-01-01

    Unlike a new generation of scientists that are being trained directly to work on the physics of living systems, most of us more senior members of the community had to find our way from other research areas. We all have our own stories as to how we made this transition. Here, I describe how a chance encounter with the eukaryotic microorganism Dictyostelium discoideum led to a decades-long research project and taught me valuable lessons about how physics and biology can be mutually supportive disciplines. PMID:25294248

  20. Learning physics of living systems from Dictyostelium

    NASA Astrophysics Data System (ADS)

    Levine, Herbert

    2014-10-01

    Unlike a new generation of scientists that are being trained directly to work on the physics of living systems, most of us more senior members of the community had to find our way from other research areas. We all have our own stories as to how we made this transition. Here, I describe how a chance encounter with the eukaryotic microorganism Dictyostelium discoideum led to a decades-long research project and taught me valuable lessons about how physics and biology can be mutually supportive disciplines.

  1. Cell motility: Combining experiments with modeling

    NASA Astrophysics Data System (ADS)

    Rappel, Wouter-Jan

    2013-03-01

    Cell migration and motility is a pervasive process in many biology systems. It involves intra-cellular signal transduction pathways that eventually lead to membrane extension and contraction. Here we describe our efforts to combine quantitative experiments with theoretical and computational modeling to gain fundamental insights into eukaryotic cell motion. In particular, we will focus on the amoeboid motion of Dictyostelium discoideum cells. This work is supported by the National Institutes of Health (P01 GM078586)

  2. Glycogen Synthase Kinase 3 influences cell motility and chemotaxis by regulating PI3K membrane localization in Dictyostelium

    PubMed Central

    Sun, Tong; Kim, Bohye; Kim, Lou W.

    2013-01-01

    Glycogen Synthase Kinase 3 (GSK3) is a multifunctional kinase involved in diverse cellular activities such as metabolism, differentiation, and morphogenesis. Recent studies showed that GSK3 in Dictyostelium affects chemotaxis via TorC2 pathway and Daydreamer. Now we report that GSK3 affects PI3K membrane localization, of which mechanism has remained to be fully understood in Dictyostelium. The membrane localization domain (LD) of Phosphatidylinositol-3-kinase 1 (PI3K1) is phosphorylated on serine residues in a GSK3 dependent mechanism and PI3K1-LD exhibited biased membrane localization in gsk3− cells compared to the wild type cells. Furthermore, multiple GSK3-phosphorylation consensus sites exist in PI3K1-LD, of which phosphomimetic substitutions restored cAMP induced transient membrane localization of PI3K1-LD in gsk3− cells. Serine to alanine substitution mutants of PI3K1-LD, in contrast, displayed constitutive membrane localization in wild type cells. Biochemical analysis revealed that GSK3 dependent serine phosphorylation of PI3K1-LD is constitutive during the course of cAMP stimulation. Together, these data suggest that GSK3 dependent serine phosphorylation is a prerequisite for chemoattractant cAMP induced PI3K membrane localization. PMID:24102085

  3. Glycogen Synthase Kinase 3 influences cell motility and chemotaxis by regulating PI3K membrane localization in Dictyostelium.

    PubMed

    Sun, Tong; Kim, Bohye; Kim, Lou W

    2013-10-01

    Glycogen Synthase Kinase 3 (GSK3) is a multifunctional kinase involved in diverse cellular activities such as metabolism, differentiation, and morphogenesis. Recent studies showed that GSK3 in Dictyostelium affects chemotaxis via TorC2 pathway and Daydreamer. Now we report that GSK3 affects PI3K membrane localization, of which the mechanism has remained to be fully understood in Dictyostelium. The membrane localization domain (LD) of Phosphatidylinositol-3-kinase 1 (PI3K1) is phosphorylated on serine residues in a GSK3 dependent mechanism and PI3K1-LD exhibited biased membrane localization in gsk3(-) cells compared to the wild type cells. Furthermore, multiple GSK3-phosphorylation consensus sites exist in PI3K1-LD, of which phosphomimetic substitutions restored cAMP induced transient membrane localization of PI3K1-LD in gsk3(-) cells. Serine to alanine substitution mutants of PI3K1-LD, in contrast, displayed constitutive membrane localization in wild type cells. Biochemical analysis revealed that GSK3 dependent serine phosphorylation of PI3K1-LD is constitutive during the course of cAMP stimulation. Together, these data suggest that GSK3 dependent serine phosphorylation is a prerequisite for chemoattractant cAMP induced PI3K membrane localization.

  4. Protein tyrosine phosphatase PTP1 negatively regulates Dictyostelium STATa and is required for proper cell-type proportioning.

    PubMed

    Early, A; Gamper, M; Moniakis, J; Kim, E; Hunter, T; Williams, J G; Firtel, R A

    2001-04-01

    The protein tyrosine phosphatase PTP1, which mediates reversible phosphorylation on tyrosine, has been shown to play an important regulatory role during Dictyostelium development. Mutants lacking PTP1 develop more rapidly than normal, while strains that overexpress PTP1 display aberrant morphology. However, the signalling pathways involved have not been characterised. In reexamining these strains, we have found that there is an inverse correlation between levels of PTP1 activity, the extent of tyrosine phosphorylation on Dictyostelium STATa after treatment with cAMP, and the proportion of the slug population exhibiting STATa nuclear enrichment in vivo. This suggests that PTP1 acts to attenuate the tyrosine phosphorylation of STATa and downstream STATa-mediated pathways. Consistent with this, we show that when PTP1 is overexpressed, there is increased expression of a prestalk cell marker at the slug posterior, a phenocopy of STATa null slugs. In ptp1 null strains, STATa tyrosine phosphorylation and nuclear enrichment in the slug anterior is increased. There is also a change in the prestalk to prespore cell ratio. Synergy experiments suggest that this is due to a cell-autonomous defect in forming the subset of prespore cells that are located in the anterior prespore region.

  5. Quantification of social behavior in D. discoideum reveals complex fixed and facultative strategies.

    PubMed

    Buttery, Neil J; Rozen, Daniel E; Wolf, Jason B; Thompson, Christopher R L

    2009-08-25

    Understanding the maintenance of cooperation requires an understanding of the nature of cheaters and the strategies used to mitigate their effects. However, it is often difficult to determine how cheating or differential social success has arisen. For example, cheaters may employ different strategies (e.g., fixed and facultative), whereas other causes of unequal fitness in social situations can result in winners and losers without cheating. To address these problems, we quantified the social success of naturally occurring genotypes of Dictyostelium discoideum during the formation of chimeric fruiting bodies, consisting of dead stalk cells and viable spores. We demonstrate that an apparent competitive dominance hierarchy of spore formation in chimera is partly due to a fixed strategy where genotypes exhibit dramatically different spore allocations. However, we also find complex, variable facultative strategies, where genotypes change their allocation in chimera. By determining the magnitude and direction of these changes, we partition facultative cheating into two forms: (1) promotion of individual fitness through selfish behaviour ("self-promotion") and (2) coercion of other genotypes to act cooperatively. Our results demonstrate and define social interactions between D. discoideum isolates, thus providing a conceptual framework for the study of the genetic mechanisms that underpin social evolution.

  6. Analysis of specific mRNA destabilization during Dictyostelium development.

    PubMed

    Mangiarotti, G; Bulfone, S; Giorda, R; Morandini, P; Ceccarelli, A; Hames, B D

    1989-07-01

    A number of specific mRNAs are destabilized upon disaggregation of developing Dictyostelium discoideum cells. Analysis of a family of cloned genes indicates that only prespore-enriched mRNAs are affected; constitutive mRNAs that are expressed throughout development and mRNAs that accumulate preferentially in prestalk cells are stable under these conditions. The decay of sensitive prespore mRNAs can be halted by allowing the cells to reaggregate, indicating that destabilization occurs by the progressive selection of individual molecules rather than on all members of an mRNA subpopulation at the time of disaggregation. Individual molecules of the sensitive mRNA species remain engaged in protein synthesis in the disaggregated cells until selected. Destabilization of sensitive mRNAs is induced by cell dissociation even in the presence of concentrations of nogalamycin that inhibit RNA synthesis. The reported prevention of disaggregation-induced mRNA decay by actinomycin D and daunomycin is therefore probably a secondary effect unrelated to the inhibition of transcription.

  7. Developmental lineage priming in Dictyostelium by heterogeneous Ras activation.

    PubMed

    Chattwood, Alex; Nagayama, Koki; Bolourani, Parvin; Harkin, Lauren; Kamjoo, Marzieh; Weeks, Gerald; Thompson, Christopher R L

    2013-11-26

    In cell culture, genetically identical cells often exhibit heterogeneous behavior, with only 'lineage primed' cells responding to differentiation inducing signals. It has recently been proposed that such heterogeneity exists during normal embryonic development to allow position independent patterning based on 'salt and pepper' differentiation and sorting out. However, the molecular basis of lineage priming and how it leads to reproducible cell type proportioning are poorly understood. To address this, we employed a novel forward genetic approach in the model organism Dictyostelium discoideum. These studies reveal that the Ras-GTPase regulator gefE is required for normal lineage priming and salt and pepper differentiation. This is because Ras-GTPase activity sets the intrinsic response threshold to lineage specific differentiation signals. Importantly, we show that although gefE expression is uniform, transcription of its target, rasD, is both heterogeneous and dynamic, thus providing a novel mechanism for heterogeneity generation and position-independent differentiation. DOI: http://dx.doi.org/10.7554/eLife.01067.001.

  8. Developmental lineage priming in Dictyostelium by heterogeneous Ras activation

    PubMed Central

    Chattwood, Alex; Nagayama, Koki; Bolourani, Parvin; Harkin, Lauren; Kamjoo, Marzieh; Weeks, Gerald; Thompson, Christopher RL

    2013-01-01

    In cell culture, genetically identical cells often exhibit heterogeneous behavior, with only ‘lineage primed’ cells responding to differentiation inducing signals. It has recently been proposed that such heterogeneity exists during normal embryonic development to allow position independent patterning based on ‘salt and pepper’ differentiation and sorting out. However, the molecular basis of lineage priming and how it leads to reproducible cell type proportioning are poorly understood. To address this, we employed a novel forward genetic approach in the model organism Dictyostelium discoideum. These studies reveal that the Ras-GTPase regulator gefE is required for normal lineage priming and salt and pepper differentiation. This is because Ras-GTPase activity sets the intrinsic response threshold to lineage specific differentiation signals. Importantly, we show that although gefE expression is uniform, transcription of its target, rasD, is both heterogeneous and dynamic, thus providing a novel mechanism for heterogeneity generation and position-independent differentiation. DOI: http://dx.doi.org/10.7554/eLife.01067.001 PMID:24282234

  9. Moving towards a paradigm: Common mechanisms of chemotactic signaling in Dictyostelium and mammalian leukocytes

    PubMed Central

    Artemenko, Yulia; Lampert, Thomas J.; Devreotes, Peter N.

    2014-01-01

    Chemotaxis, or directed migration of cells along a chemical gradient, is a highly coordinated process that involves gradient sensing, motility, and polarity. Most of our understanding of chemotaxis comes from studies of cells undergoing amoeboid-type migration, in particular the social amoeba Dictyostelium discoideum and leukocytes. In these amoeboid cells the molecular events leading to directed migration can be conceptually divided into four interacting networks: receptor/G protein, signal transduction, cytoskeleton, and polarity. The signal transduction network occupies a central position in this scheme as it receives direct input from the receptor/G protein network, as well as feedback from the cytoskeletal and polarity networks. Multiple overlapping modules within the signal transduction network transmit the signals to the actin cytoskeleton network leading to biased pseudopod protrusion in the direction of the gradient. The overall architecture of the networks, as well as the individual signaling modules are remarkably conserved between Dictyostelium and mammalian leukocytes, and the similarities and differences between the two systems are the subject of this review. PMID:24846395

  10. RasG signaling is important for optimal folate chemotaxis in Dictyostelium

    PubMed Central

    2014-01-01

    Background Signaling pathways linking receptor activation to actin reorganization and pseudopod dynamics during chemotaxis are arranged in complex networks. Dictyostelium discoideum has proven to be an excellent model system for studying these networks and a body of evidence has indicated that RasG and RasC, members of the Ras GTPase subfamily function as key chemotaxis regulators. However, recent evidence has been presented indicating that Ras signaling is not important for Dictyostelium chemotaxis. In this study, we have reexamined the role of Ras proteins in folate chemotaxis and then, having re-established the importance of Ras for this process, identified the parts of the RasG protein molecule that are involved. Results A direct comparison of folate chemotaxis methodologies revealed that rasG-C- cells grown in association with a bacterial food source were capable of positive chemotaxis, only when their initial position was comparatively close to the folate source. In contrast, cells grown in axenic medium orientate randomly regardless of their distance to the micropipette. Folate chemotaxis is restored in rasG-C- cells by exogenous expression of protein chimeras containing either N- or C- terminal halves of the RasG protein. Conclusions Conflicting data regarding the importance of Ras to Dictyostelium chemotaxis were the result of differing experimental methodologies. Both axenic and bacterially grown cells require RasG for optimal folate chemotaxis, particularly in weak gradients. In strong gradients, the requirement for RasG is relaxed, but only in bacterially grown cells. Both N- and C- terminal portions of the RasG protein are important for folate chemotaxis, suggesting that there are functionally important amino acids outside the well established switch I and switch II interaction surfaces. PMID:24742374

  11. A Dictyostelium mutant lacking an F-actin cross-linking protein, the 120-kD gelation factor

    PubMed Central

    1990-01-01

    Actin-binding proteins are known to regulate in vitro the assembly of actin into supramolecular structures, but evidence for their activities in living nonmuscle cells is scarce. Amebae of Dictyostelium discoideum are nonmuscle cells in which mutants defective in several actin-binding proteins have been described. Here we characterize a mutant deficient in the 120-kD gelation factor, one of the most abundant F-actin cross- linking proteins of D. discoideum cells. No F-actin cross-linking activity attributable to the 120-kD protein was detected in mutant cell extracts, and antibodies recognizing different epitopes on the polypeptide showed the entire protein was lacking. Under the conditions used, elimination of the gelation factor did not substantially alter growth, shape, motility, or chemotactic orientation of the cells towards a cAMP source. Aggregates of the mutant developed into fruiting bodies consisting of normally differentiated spores and stalk cells. In cytoskeleton preparations a dense network of actin filaments as typical of the cell cortex, and bundles as they extend along the axis of filopods, were recognized. A significant alteration found was an enhanced accumulation of actin in cytoskeletons of the mutant when cells were stimulated with cyclic AMP. Our results indicate that control of cell shape and motility does not require the fine-tuned interactions of all proteins that have been identified as actin-binding proteins by in vitro assays. PMID:1698791

  12. Dual chemotaxis signalling regulates Dictyostelium development: intercellular cyclic AMP pulses and intracellular F-actin disassembly waves induce each other.

    PubMed

    Vicker, Michael G; Grutsch, James F

    2008-10-01

    Aggregating Dictyostelium discoideum amoebae periodically emit and relay cAMP, which regulates their chemotaxis and morphogenesis into a multicellular, differentiated organism. Cyclic AMP also stimulates F-actin assembly and chemotactic pseudopodium extension. We used actin-GFP expression to visualise for the first time intracellular F-actin assembly as a spatio-temporal indicator of cell reactions to cAMP, and thus the kinematics of cell communication, in aggregating streams. Every natural cAMP signal pulse induces an autowave of F-actin disassembly, which propagates from each cell's leading end to its trailing end at a linear rate, much slower than the calculated and measured velocities of cAMP diffusion in aggregating Dictyostelium. A sequence of transient reactions follows behind the wave, including anterior F-actin assembly, chemotactic pseudopodium extension and cell advance at the cell front and, at the back, F-actin assembly, extension of a small retrograde pseudopodium (forcing a brief cell retreat) and chemotactic stimulation of the following cell, yielding a 20s cAMP relay delay. These dynamics indicate that stream cell behaviour is mediated by a dual signalling system: a short-range cAMP pulse directed from one cell tail to an immediately following cell front and a slower, long-range wave of intracellular F-actin disassembly, each inducing the other.

  13. A Dictyostelium cellobiohydrolase orthologue that affects developmental timing.

    PubMed

    Kunii, Mizuho; Yasuno, Mami; Shindo, Yuki; Kawata, Takefumi

    2014-02-01

    Dictyostelium discoideum is a facultative multicellular amoebozoan with cellulose in the stalk and spore coat of its fruiting body as well as in the extracellular matrix of the migrating slug. The organism also harbors a number of cellulase genes. One of them, cbhA, was identified as a candidate cellobiohydrolase gene based on the strong homology of its predicted protein product to fungal cellobiohydrolase I (CBHI). Expression of the cbhA was developmentally regulated, with strong expression in the spores of the mature fruiting body. However, a weak but detectable level of expression was observed in the extracellular matrix at the mound - tipped finger stages, in prestalk O cells, and in the slime sheath of the migrating slug - late culminant stages. A null mutant of the cbhA showed almost normal morphology. However, the developmental timing of the mutant was delayed by 2-4 h. When a c-Myc epitope-tagged CbhA was expressed, it was secreted into the culture medium and was able to bind crystalline cellulose. The CbhA-myc protein was glycosylated, as demonstrated by its ability to bind succinyl concanavalin A-agarose. Moreover, conditioned medium from the cbhA-myc (oe) strain displayed 4-methylumbelliferyl β-D-cellobioside (4-MUC) digesting activity in Zymograms in which conditioned medium was examined via native-polyacrylamide gel electrophoresis or spotted on an agar plate containing 4-MUC, one of the substrates of cellobiohydrolase. Taken together, these findings indicate that Dictyostelium CbhA is an orthologue of CBH I that is required for a normal rate of development.

  14. The GATA transcription factor GtaC regulates early developmental gene expression dynamics in Dictyostelium.

    PubMed

    Santhanam, Balaji; Cai, Huaqing; Devreotes, Peter N; Shaulsky, Gad; Katoh-Kurasawa, Mariko

    2015-07-06

    In many systems, including the social amoeba Dictyostelium discoideum, development is often marked by dynamic morphological and transcriptional changes orchestrated by key transcription factors. However, efforts to examine sequential genome-wide changes of gene regulation in developmental processes have been fairly limited. Here we report the developmental regulatory dynamics of GtaC, a GATA-type zinc-finger transcription factor, through the analyses of serial ChIP- and RNA-sequencing data. GtaC is essential for developmental progression, decoding extracellular cAMP pulses during early development and may play a role in mediating cell-type differentiation at later stages. We find that GtaC exhibits temporally distinctive DNA-binding patterns concordant with each developmental stage. We identify direct GtaC targets and observe cotemporaneous GtaC-binding and developmental expression regulation. Our results suggest that GtaC regulates multiple physiological processes as Dictyostelium transitions from a group of unicellular amoebae to an integrated multicellular organism.

  15. Xpf suppresses the mutagenic consequences of phagocytosis in Dictyostelium

    PubMed Central

    Langenick, Judith; Zhang, Xiao-Yin; Traynor, David; Kay, Robert R.

    2016-01-01

    ABSTRACT As time passes, mutations accumulate in the genomes of all living organisms. These changes promote genetic diversity, but also precipitate ageing and the initiation of cancer. Food is a common source of mutagens, but little is known about how nutritional factors cause lasting genetic changes in the consuming organism. Here, we describe an unusual genetic interaction between DNA repair in the unicellular amoeba Dictyostelium discoideum and its natural bacterial food source. We found that Dictyostelium deficient in the DNA repair nuclease Xpf (xpf−) display a severe and specific growth defect when feeding on bacteria. Despite being proficient in the phagocytosis and digestion of bacteria, over time, xpf− Dictyostelium feeding on bacteria cease to grow and in many instances die. The Xpf nuclease activity is required for sustained growth using a bacterial food source. Furthermore, the ingestion of this food source leads to a striking accumulation of mutations in the genome of xpf− Dictyostelium. This work therefore establishes Dictyostelium as a model genetic system to dissect nutritional genotoxicity, providing insight into how phagocytosis can induce mutagenesis and compromise survival fitness. PMID:27872153

  16. Dictyostelium ACAP-A is an ArfGAP involved in cytokinesis, cell migration and actin cytoskeleton dynamics.

    PubMed

    Dias, Marco; Blanc, Cédric; Thazar-Poulot, Nelcy; Ben Larbi, Sabrina; Cosson, Pierre; Letourneur, François

    2013-02-01

    ACAPs and ASAPs are Arf-GTPase-activating proteins with BAR, PH, GAP and ankyrin repeat domains and are known to regulate vesicular traffic and actin cytoskeleton dynamics in mammalian cells. The amoeba Dictyostelium has only two proteins with this domain organization, instead of the six in human, enabling a more precise functional analysis. Genetic invalidation of acapA resulted in multinucleated cells with cytokinesis defects. Mutant acapA(-) cells were hardly motile and their multicellular development was significantly delayed. In addition, formation of filopodial protrusions was deficient in these cells. Conversely, re-expression of ACAP-A-GFP resulted in numerous and long filopodia-like protrusions. Mutagenesis studies showed that the ACAP-A actin remodeling function was dependent on its ability to activate its substrate, the small GTPase ArfA. Likewise, the expression of a constitutively active ArfA•GTP mutant in wild-type cells led to a significant reduction in filopodia length. Together, our data support a role for ACAP-A in the control of the actin cytoskeleton organization and dynamics through an ArfA-dependent mechanism.

  17. ACAP-A/B are ArfGAP homologs in dictyostelium involved in sporulation but not in chemotaxis.

    PubMed

    Chen, Pei-Wen; Randazzo, Paul A; Parent, Carole A

    2010-01-07

    Arfs and Arf GTPase-activating proteins (ArfGAPs) are regulators of membrane trafficking and actin dynamics in mammalian cells. In this study, we identified a primordial Arf, ArfA, and two ArfGAPs (ACAP-A/B) containing BAR, PH, ArfGAP and Ankyrin repeat domains in the eukaryote Dictyostelium discoideum. In vitro, ArfA has similar nucleotide binding properties as mammalian Arfs and, with GTP bound, is a substrate for ACAP-A and B. We also investigated the physiological functions of ACAP-A/B by characterizing cells lacking both ACAP-A and B. Although ACAP-A/B knockout cells showed no defects in cell growth, migration or chemotaxis, they exhibited abnormal actin protrusions and approximately 50% reduction in spore yield. We conclude that while ACAP-A/B have a conserved biochemical mechanism and effect on actin organization, their role in migration is not conserved. The absence of an effect on Dictyostelium migration may be due to a specific requirement for ACAPs in mesenchymal migration, which is observed in epithelial cancer cells where most studies of mammalian ArfGAPs were performed.

  18. Pattern formation of Dictystelium discoideum in the presence of laminar flow and cAMP pulses

    NASA Astrophysics Data System (ADS)

    Gholami, Azam; Steinbock, Oliver; Zykov, Vladimir; Bodenschatz, Eberhard

    2014-03-01

    Dictyostelium discoideum (D.d) amobae undergo starvation-induced multicellular development in which single cells aggregate chemotactically towards cAMP signals emitted periodically from an aggregation center. We are investigating spatiotemporal pattern formation of D.d. cells under the presence of a laminar flow. Starved cells are loaded into a straight millifluidic device with an external flow and cell response to the signaling molecule cAMP is monitored indirectly using dark-field microscopy. The observed contraction waves develop simultaneously over the entire channel, are propagating only in flow direction, and have curved wave fronts resembling the parabolic flow profile. The wave dynamics analysis shows that the wave velocity is locked to the flow velocity and yields a wave period of T0 6 min, which matches the typical oscillation period of extracellular cAMP in spatial homogeneous, well-stirred systems. We apply a small cAMP perturbation at the inlet region of the channel and observe the spatiotemporal response of the cells as the pulse is propagating down the channel. The results show that D.d. cells are in the oscillatory regime and the system can be forced within resonance tongue. We compared our results with analytical and numerical analysis of Goldbeter model.

  19. lagC-null and gbf-null cells define key steps in the morphogenesis of Dictyostelium mounds.

    PubMed

    Sukumaran, S; Brown, J M; Firtel, R A; McNally, J G

    1998-08-01

    The transition to multicellularity is a key feature of the Dictyostelium life cycle, and two genes, gbf and lagC, are known to play pivotal roles in regulating this developmental switch. lagC-null and gbf-null cells fail to induce cell-type-specific genes ordinarily expressed during multicellular development. The null mutants also share a similar morphological phenotype: mutant cells repeatedly aggregate to form a loose mound, disperse, and reform a mound, rather than proceeding to form a tip. To characterize defects in morphogenesis in these mutants, we examined cell motion in the mutant mounds. In analogy with the failed transition in gene expression, we found that lagC-null and gbf-null mounds failed to make a morphogenetic transition from random to rotational motion normally observed in the parent strain. One reason for this was the inability of the mutant mounds to establish a single, dominant signaling-wave center. This defect of lagC-null or gbf-null cells could be overcome by the addition of adenosine, which alters cAMP signaling, but then even in the presence of apparently normal signaling waves, cell motility was still aberrant. This motility defect, as well as the signaling-wave defect, could be overcome in lagC-null cells by overexpression of GBF, suggesting that lagC is dispensable if GBF protein levels are high enough. This set of morphogenetic defects that we have observed helps define key steps in mound morphogenesis. These include the establishment of a dominant signaling-wave center and the capacity of cells to move directionally within the cell mass in response to guidance cues.

  20. Identification of a Suppressor of the Dictyostelium Profilin-minus Phenotype as a CD36/LIMP-II Homologue

    PubMed Central

    Karakesisoglou, Iakowos; Janssen, Klaus-Peter; Eichinger, Ludwig; Noegel, Angelika A.; Schleicher, Michael

    1999-01-01

    Profilin is an ubiquitous G-actin binding protein in eukaryotic cells. Lack of both profilin isoforms in Dictyostelium discoideum resulted in impaired cytokinesis and an arrest in development. A restriction enzyme–mediated integration approach was applied to profilin-minus cells to identify suppressor mutants for the developmental phenotype. A mutant with wild-type–like development and restored cytokinesis was isolated. The gene affected was found to code for an integral membrane glycoprotein of a predicted size of 88 kD containing two transmembrane domains, one at the NH2 terminus and the other at the COOH terminus. It is homologous to mammalian CD36/LIMP-II and represents the first member of this family in D. discoideum, therefore the name DdLIMP is proposed. Targeted disruption of the lmpA gene in the profilin-minus background also rescued the mutant phenotype. Immunofluorescence revealed a localization in vesicles and ringlike structures on the cell surface. Partially purified DdLIMP bound specifically to PIP2 in sedimentation and gel filtration assays. A direct interaction between DdLIMP and profilin could not be detected, and it is unclear how far upstream in a regulatory cascade DdLIMP might be positioned. However, the PIP2 binding of DdLIMP points towards a function via the phosphatidylinositol pathway, a major regulator of profilin. PMID:10189376

  1. Skipper, an LTR retrotransposon of Dictyostelium.

    PubMed Central

    Leng, P; Klatte, D H; Schumann, G; Boeke, J D; Steck, T L

    1998-01-01

    The complete sequence of a retrotransposon from Dictyostelium discoideum , named skipper , was obtained from cDNA and genomic clones. The sequence of a nearly full-length skipper cDNA was similar to that of three other partially sequenced cDNAs. The corresponding retrotransposon is represented in approximately 15-20 copies and is abundantly transcribed. Skipper contains three open reading frames (ORFs) with an unusual sequence organization, aspects of which resemble certain mammalian retroviruses. ORFs 1 and 3 correspond to gag and pol genes; the second ORF, pro, corresponding to protease, was separated from gag by a single stop codon followed shortly thereafter by a potential pseudoknot. ORF3 (pol) was separated from pro by a +1 frameshift. ORFs 2 and 3 overlapped by 32 bp. The computed amino acid sequences of the skipper ORFs contain regions resembling retrotransposon polyprotein domains, including a nucleic acid binding protein, aspartyl protease, reverse transcriptase and integrase. Skipper is the first example of a retrotransposon with a separate pro gene. Skipper is also novel in that it appears to use stop codon suppression rather than frameshifting to modulate pro expression. Finally, skipper and its components may provide useful tools for the genetic characterization of Dictyostelium. PMID:9518497

  2. Morphogenesis, Dictyostelium, and the search for shared developmental processes.

    PubMed

    Sunderland, Mary Evelyn

    2011-12-01

    In the 1930s John Tyler Bonner began studying the slime mold, Dictyostelium discoideum, as a way to investigate how organisms develop. With a life cycle that includes periods of unicellularity and multicellularity, Dictyostelium raises questions fundamental to development and evolution. In Morphogenesis: An Essay on Development (1952), Bonner built on his work with Dictyostelium to inform developmental theory and practice. By exploring how Bonner's early work with Dictyostelium motivated his synthetic approach in Morphogenesis, this paper presents an example of how those who studied development sought ways to gain traction in the rapidly changing life sciences. While a biochemical viewpoint of development became dominant, morphogenesis provided a way to reintroduce and emphasize biological organization at the organismal level. Bonner's early work offers a window to mid-twentieth century studies of development, an understudied area in the history of science, and shows that it was a time when growing experimental evidence enabled new ways of thinking about the relationship between ontogeny and evolution, and more broadly, about how the parts of nature might fit together.

  3. EGF-like peptide-enhanced cell movement in Dictyostelium is mediated by protein kinases and the activity of several cytoskeletal proteins.

    PubMed

    Huber, Robert J; O'Day, Danton H

    2012-09-01

    DdEGFL1, a synthetic epidermal growth factor-like (EGFL) peptide based on the first EGFL repeat of the extracellular matrix, cysteine-rich, calmodulin-binding protein CyrA, has previously been shown to sustain the threonine phosphorylation of a 210kDa protein during the starvation of Dictyostelium cells. Immunoprecipitation coupled with a LC/MS/MS analysis identified the 210kDa protein as vinculin B (VinB). VinB shares sequence similarity with mammalian vinculin, a protein that links the actin cytoskeleton to the plasma membrane. Both threonine phosphorylated VinB (P-VinB) and VinB-GFP localized to the cytoplasm and cytoskeleton of Dictyostelium amoebae. VinB-GFP was also shown to be threonine phosphorylated and co-immunoprecipitated with established vinculin-binding cytoskeletal proteins (e.g. myosin II heavy chain, actin, alpha-actinin, talin). P-VinB and VinB-GFP were detected in DdEGFL1 pull-down assays, which also identified a 135kDa phosphothreonine protein and two phosphotyrosine proteins (35 and 32kDa) as potential components of the DdEGFL1 signaling pathway. DdEGFL1-enhanced cell movement required the cytoskeletal proteins talin B and paxillin B and tyrosine kinase activity mediated by PKA signaling, however VinB threonine phosphorylation was shown to be independent of PI3K/PLA2 signaling and PI3K and PKA kinase activity. Finally, VinB-GFP over-expression suppressed DdEGFL1-enhanced random cell movement, but not folic acid-mediated chemotaxis. Together, this study provides the first evidence for VinB function plus new insight into the signaling pathway(s) mediating EGFL repeat/peptide-enhanced cell movement in Dictyostelium. This information is integrated into an emerging model that summarizes existing knowledge.

  4. The GATA transcription factor gene gtaG is required for terminal differentiation in Dictyostelium.

    PubMed

    Katoh-Kurasawa, Mariko; Santhanam, Balaji; Shaulsky, Gad

    2016-03-09

    The GATA transcription factor GtaG is conserved in Dictyostelids and essential for terminal differentiation in Dictyostelium discoideum, but its function is not well understood. Here we show that gtaG is expressed in prestalk cells at the anterior region of fingers and in the extending stalk during culmination. The gtaG(-) phenotype is cell-autonomous in prestalk cells and non-cell-autonomous in prespore cells. Transcriptome analyses reveal that GtaG regulates prestalk gene expression during cell differentiation before culmination and is required for progression into culmination. GtaG-dependent genes include genetic suppressors of the Dd-STATa-defective phenotype as well as Dd-STATa target-genes, including extra cellular matrix genes. We show that GtaG may be involved in the production of two culmination-signaling molecules, cyclic di-GMP and the spore differentiation factor SDF-1 and that addition of c-di-GMP rescues the gtaG(-) culmination and spore formation deficiencies. We propose that GtaG is a regulator of terminal differentiation that functions in concert with Dd-STATa and controls culmination through regulating c-di-GMP and SDF-1 production in prestalk cells.

  5. ForC lacks canonical formin activity but bundles actin filaments and is required for multicellular development of Dictyostelium cells.

    PubMed

    Junemann, Alexander; Winterhoff, Moritz; Nordholz, Benjamin; Rottner, Klemens; Eichinger, Ludwig; Gräf, Ralph; Faix, Jan

    2013-01-01

    Diaphanous-related formins (DRFs) drive the nucleation and elongation of linear actin filaments downstream of Rho GTPase signalling pathways. Dictyostelium formin C (ForC) resembles a DRF, except that it lacks a genuine formin homology domain 1 (FH1), raising the questions whether or not ForC can nucleate and elongate actin filaments. We found that a recombinant ForC-FH2 fragment does not nucleate actin polymerization, but moderately decreases the rate of spontaneous actin assembly and disassembly, although the barbed-end elongation rate in the presence of the formin was not markedly changed. However, the protein bound to and crosslinked actin filaments into loose bundles of mixed polarity. Furthermore, ForC is an important regulator of morphogenesis since ForC-null cells are severely impaired in development resulting in the formation of aberrant fruiting bodies. Immunoblotting revealed that ForC is absent during growth, but becomes detectable at the onset of early aggregation when cells chemotactically stream together to form a multicellular organism, and peaks around the culmination stage. Fluorescence microscopy of cells ectopically expressing a GFP-tagged, N-terminal ForC fragment showed its prominent accumulation in the leading edge, suggesting that ForC may play a role in cell migration. In agreement with its expression profile, no defects were observed in random migration of vegetative mutant cells. Notably, chemotaxis of starved cells towards a source of cAMP was severely impaired as opposed to control. This was, however, largely due to a marked developmental delay of the mutant, as evidenced by the expression profile of the early developmental marker csA. In line with this, chemotaxis was almost restored to wild type levels after prolonged starvation. Finally, we observed a complete failure of phototaxis due to abolished slug formation and a massive reduction of spores consistent with forC promoter-driven expression of β-galactosidase in prespore cells

  6. Nse1 and Nse4, subunits of the Smc5-Smc6 complex, are involved in Dictyostelium development upon starvation.

    PubMed

    Taniura, Hideo; Tanabe, Naoya; Bando, Yumi; Arai, Natsumi

    2015-08-01

    The Smc5-Smc6 complex contains a heterodimeric core of two SMC proteins and non-Smc elements (Nse1-6), and plays an important role in DNA repair. We investigated the functional roles of Nse4 and Nse1 in Dictyostelium discoideum. Nse4 and Nse3 expressed as Flag-tagged fusion proteins were highly enriched in nuclei, while Nse1 was localized in whole cells. Using yeast two-hybrid assays, only the interaction between Nse3 and Nse1 was detected among the combinations. However, all of the interactions among these three proteins were recognized by co-immunoprecipitation assay using cell lysates prepared from the cells expressing green fluorescent protein (GFP)- or Flag-tagged fusion proteins. GFP-tagged Nse1, which localized in whole cells, was translocated to nuclei when co-expressed with Flag-tagged Nse3 or Nse4. RNAi-mediated Nse1 and Nse4 knockdown cells (Nse1 KD and Nse4 KD cells) were generated and found to be more sensitive to UV-induced cell death than control cells. Upon starvation, Nse1 and Nse4 KD cells had increases in the number of smaller fruiting bodies that formed on non-nutrient agar plates or aggregates that formed under submerged culture. We found a reduction in the mRNA level of pdsA, in vegetative and 8 h-starved Nse4 KD cells, and pdsA knockdown cells displayed effects similar to Nse4 KD cells. Our results suggest that Nse4 and Nse1 are involved in not only the cellular DNA damage response but also cellular development in D. discoideum.

  7. Addition and correction: the NF-kappa B-like DNA binding activity observed in Dictyostelium nuclear extracts is due to the GBF transcription factor.

    PubMed

    Traincard, F; Ponte, E; Pun, J; Coukell, B; Veron, M

    2001-10-01

    We have previously reported that a NF-kappa B transduction pathway was likely to be present in the cellular slime mold Dictyostelium discoideum. This conclusion was based on several observations, including the detection of developmentally regulated DNA binding proteins in Dictyostelium nuclear extracts that bound to bona fide kappa B sequences. We have now performed additional experiments which demonstrate that the protein responsible for this NF-kappa B-like DNA binding activity is the Dictyostelium GBF (G box regulatory element binding factor) transcription factor. This result, along with the fact that no sequence with significant similarity to components of the mammalian NF-kappa B pathway can be found in Dictyostelium genome, now almost entirely sequenced, led us to reconsider our previous conclusion on the occurrence of a NF-kappa B signal transduction pathway in Dictyostelium.

  8. F-actin assembly in Dictyostelium cell locomotion and shape oscillations propagates as a self-organized reaction-diffusion wave.

    PubMed

    Vicker, Michael G

    2002-01-02

    The crawling locomotion and shape of eukaryotic cells have been associated with the stochastic molecular dynamics of actin and its protein regulators, chiefly Arp2/3 and Rho family GTPases, in making a cytoskeleton meshwork within cell extensions. However, the cell's actin-dependent oscillatory shape and extension dynamics may also yield insights into locomotory mechanisms. Confocal observations of live Dictyostelium cells, expressing a green fluorescent protein-actin fusion protein, demonstrate oscillating supramolecular patterns of filamentous actin throughout the cell, which generate pseudopodia at the cell edge. The distinctively dissipative spatio-temporal behavior of these structures provides strong evidence that reversible actin filament assembly propagates as a self-organized, chemical reaction-diffusion wave.

  9. One stop shop for everything Dictyostelium: dictyBase and the Dicty Stock Center in 2012

    PubMed Central

    Fey, Petra; Dodson, Robert J.; Basu, Siddhartha; Chisholm, Rex L.

    2013-01-01

    dictyBase (http:// dictybase.org), the model organism database for Dictyostelium discoideum, includes the complete genome sequence and expression data for this organism. Relevant literature is integrated into the database, and gene models and functional annotation are manually curated from experimental results and comparative multigenome analyses. dictyBase has recently expanded to include the genome sequences of three additional Dictyostelids, and has added new software tools to facilitate multigenome comparisons. The Dicty Stock Center, a strain and plasmid repository for Dictyostelium research has relocated to Northwestern University in 2009. This allowed us integrating all Dictyostelium resources to better serve the research community. In this chapter, we will describe how to navigate the website and highlight some of our newer improvements. PMID:23494302

  10. Controlling Collective Behaviors of Dictyostelium

    NASA Astrophysics Data System (ADS)

    Schwab, David; Mehta, Pankaj; Gregor, Thomas

    2010-03-01

    We study the collective dynamics of a population of Dictyostelium cells, focusing on how single cell dynamics influence, and give rise to, the behavior of the aggregate. Through analysis of quantitative single cell experiments, we develop a simple model of the single cell response to time-dependent pulses of the extracellular signaling molecule cAMP, characterized by a particular type of excitable system. We then use this model to study collective multicellular dynamics mediated by diffusion coupling. We first consider the mean-field case where we find an intriguing ``dynamical quorum sensing'' transition in which all cells simultaneously transition from quiescent to oscillating across the phase boundary. Then we include spatial dynamics and study pattern formation, both with and without the cells capable of chemotactic response to signal gradients. Finally, we highlight how modification of single cells can alter the collective dynamics.

  11. Adhesion of D. discoideum on Hydrophobic Substrate

    NASA Astrophysics Data System (ADS)

    Flanders, Bret; Ploscariu, Nicoleta

    2015-03-01

    Adhesion by amoeboid cells, such as D. discoideum, is poorly understood but critical for other behaviors such as phagocytosis and migration. Furthermore, both leucocytes and breast cancer cells employ the amoeboid mode of movement at various points in their life-cycles. Hence, improved knowledge of amoeboid adhesion may lead to be new strategies for controlling other important cellular processes. This study regards adhesion by D. discoideum on silanized glass substrates. Reflection interference contrast microscopy is used in conjunction with other methods to determine the contact angle, cell-medium interfacial energy, and adhesion energy of these cells. The contact angle of individual cells settling under gravity onto a substrate is observed to increase as the size of the contact patch increases. This behavior occurs on slower time-scales than expected for the settling of inert vesicles. The implications of this observation on the nature of the underlying forces will be discussed. This work was supported in part by NSF Grant PHY-646966.

  12. Autophagy in Dictyostelium: Mechanisms, regulation and disease in a simple biomedical model.

    PubMed

    Mesquita, Ana; Cardenal-Muñoz, Elena; Dominguez, Eunice; Muñoz-Braceras, Sandra; Nuñez-Corcuera, Beatriz; Phillips, Ben A; Tábara, Luis C; Xiong, Qiuhong; Coria, Roberto; Eichinger, Ludwig; Golstein, Pierre; King, Jason S; Soldati, Thierry; Vincent, Olivier; Escalante, Ricardo

    2017-01-02

    Autophagy is a fast-moving field with an enormous impact on human health and disease. Understanding the complexity of the mechanism and regulation of this process often benefits from the use of simple experimental models such as the social amoeba Dictyostelium discoideum. Since the publication of the first review describing the potential of D. discoideum in autophagy, significant advances have been made that demonstrate both the experimental advantages and interest in using this model. Since our previous review, research in D. discoideum has shed light on the mechanisms that regulate autophagosome formation and contributed significantly to the study of autophagy-related pathologies. Here, we review these advances, as well as the current techniques to monitor autophagy in D. discoideum. The comprehensive bioinformatics search of autophagic proteins that was a substantial part of the previous review has not been revisited here except for those aspects that challenged previous predictions such as the composition of the Atg1 complex. In recent years our understanding of, and ability to investigate, autophagy in D. discoideum has evolved significantly and will surely enable and accelerate future research using this model.

  13. PakB binds to the SH3 domain of Dictyostelium Abp1 and regulates its effects on cell polarity and early development.

    PubMed

    Yang, Yidai; de la Roche, Marc; Crawley, Scott W; Li, Zhihao; Furmaniak-Kazmierczak, Emilia; Côté, Graham P

    2013-07-01

    Dictyostelium p21-activated kinase B (PakB) phosphorylates and activates class I myosins. PakB colocalizes with myosin I to actin-rich regions of the cell, including macropinocytic and phagocytic cups and the leading edge of migrating cells. Here we show that residues 1-180 mediate the cellular localization of PakB. Yeast two-hybrid and pull-down experiments identify two proline-rich motifs in PakB-1-180 that directly interact with the SH3 domain of Dictyostelium actin-binding protein 1 (dAbp1). dAbp1 colocalizes with PakB to actin-rich regions in the cell. The loss of dAbp1 does not affect the cellular distribution of PakB, whereas the loss of PakB causes dAbp1 to adopt a diffuse cytosolic distribution. Cosedimentation studies show that the N-terminal region of PakB (residues 1-70) binds directly to actin filaments, whereas dAbp1 exhibits only a low affinity for filamentous actin. PakB-1-180 significantly enhances the binding of dAbp1 to actin filaments. When overexpressed in PakB-null cells, dAbp1 completely blocks early development at the aggregation stage, prevents cell polarization, and significantly reduces chemotaxis rates. The inhibitory effects are abrogated by the introduction of a function-blocking mutation into the dAbp1 SH3 domain. We conclude that PakB plays a critical role in regulating the cellular functions of dAbp1, which are mediated largely by its SH3 domain.

  14. Organization of microtubule assemblies in Dictyostelium syncytia depends on the microtubule crosslinker, Ase1.

    PubMed

    Tikhonenko, Irina; Irizarry, Karen; Khodjakov, Alexey; Koonce, Michael P

    2016-02-01

    It has long been known that the interphase microtubule (MT) array is a key cellular scaffold that provides structural support and directs organelle trafficking in eukaryotic cells. Although in animal cells, a combination of centrosome nucleating properties and polymer dynamics at the distal microtubule ends is generally sufficient to establish a radial, polar array of MTs, little is known about how effector proteins (motors and crosslinkers) are coordinated to produce the diversity of interphase MT array morphologies found in nature. This diversity is particularly important in multinucleated environments where multiple MT arrays must coexist and function. We initiate here a study to address the higher ordered coordination of multiple, independent MT arrays in a common cytoplasm. Deletion of a MT crosslinker of the MAP65/Ase1/PRC1 family disrupts the spatial integrity of multiple arrays in Dictyostelium discoideum, reducing the distance between centrosomes and increasing the intermingling of MTs with opposite polarity. This result, coupled with previous dynein disruptions suggest a robust mechanism by which interphase MT arrays can utilize motors and crosslinkers to sense their position and minimize overlap in a common cytoplasm.

  15. Organization of microtubule assemblies in Dictyostelium syncytia depends on the microtubule crosslinker, Ase1

    PubMed Central

    Tikhonenko, Irina; Irizarry, Karen; Khodjakov, Alexey; Koonce, Michael P.

    2015-01-01

    It has long been known that the interphase microtubule (MT) array is a key cellular scaffold that provides structural support and directs organelle trafficking in eukaryotic cells. Although in animal cells, a combination of centrosome nucleating properties and polymer dynamics at the distal microtubule ends is generally sufficient to establish a radial, polar array of MTs, little is known about how effector proteins (motors and crosslinkers) are coordinated to produce the diversity of interphase MT array morphologies found in nature. This diversity is particularly important in multinucleated environments where multiple MT arrays must coexist and function. We initiate here a study to address the higher ordered coordination of multiple, independent MT arrays in a common cytoplasm. Deletion of a MT crosslinker of the MAP65/Ase1/PRC1 family disrupts the spatial integrity of multiple arrays in Dictyostelium discoideum, reducing the distance between centrosomes and increasing the intermingling of MTs with opposite polarity. This result, coupled with previous dynein disruptions suggest a robust mechanism by which interphase MT arrays can utilize motors and crosslinkers to sense their position and minimize overlap in a common cytoplasm. PMID:26298292

  16. A Dictyostelium mutant deficient in severin, an F-actin fragmenting protein, shows normal motility and chemotaxis

    PubMed Central

    1989-01-01

    A severin deficient mutant of Dictyostelium discoideum has been isolated by the use of colony immunoblotting after chemical mutagenesis. In homogenates of wild-type cells, severin is easily detected as a very active F-actin fragmenting protein. Tests for severin in the mutant, HG1132, included viscometry for the assay of F- actin fragmentation in fractions from DEAE-cellulose columns, labeling of blots with monoclonal and polyclonal antibodies, and immunofluorescent-labeling of cryosections. Severin could not be detected in the mutant using these methods. The mutation in HG1132 is recessive and has been mapped to linkage group VII. The mutant failed to produce the normal severin mRNA, but small amounts of a transcript that was approximately 100 bases larger than the wild-type mRNA were detected in the mutant throughout all stages of development. On the DNA level a new Mbo II restriction site was found in the mutant within the coding region of the severin gene. The severin deficient mutant cells grew at an approximately normal rate, aggregated and formed fruiting bodies with viable spores. By the use of an image processing system, speed of cell movement, turning rates, and precision of chemotactic orientation in a stable gradient of cyclic AMP were quantitated, and no significant differences between wild-type and mutant cells were found. Thus, under the culture conditions used, severin proved to be neither essential for growth of D. discoideum nor for any cell function that is important for aggregation or later development. PMID:2537840

  17. An evolutionarily significant unicellular strategy in response to starvation in Dictyostelium social amoebae

    PubMed Central

    Dubravcic, Darja; van Baalen, Minus; Nizak, Clément

    2014-01-01

    The social amoeba Dictyostelium discoideum is widely studied for its multicellular development program as a response to starvation. Aggregates of up to 10 6 cells form fruiting bodies containing (i) dormant spores (~80%) that can persist for months in the absence of nutrients, and (ii) dead stalk cells (~20%) that promote the dispersion of the spores towards nutrient-rich areas. It is often overlooked that not all cells aggregate upon starvation. Using a new quantitative approach based on time-lapse fluorescence microscopy and a low ratio of reporting cells, we have quantified this fraction of non-aggregating cells. In realistic starvation conditions, up to 15% of cells do not aggregate, which makes this third cell fate a significant component of the population-level response of social amoebae to starvation. Non-aggregating cells have an advantage over cells in aggregates since they resume growth earlier upon arrival of new nutrients, but have a shorter lifespan under prolonged starvation. We find that phenotypic heterogeneities linked to cell nutritional state bias the representation of cells in the aggregating vs. non-aggregating fractions, and thus affect population partitioning. Next, we report that the fraction of non-aggregating cells depends on genetic factors that regulate the timing of starvation, signal sensing efficiency and aggregation efficiency. In addition, interactions between clones in mixtures of non-isogenic cells affect the partitioning of each clone into both fractions. We further build a numerical model to test the evolutionary significance of the non-aggregating cell fraction. The partitioning of cells into aggregating and non-aggregating fractions is optimal in fluctuating environments with an unpredictable duration of starvation periods. Our study highlights the unicellular component of the response of social amoebae to starvation, and thus extends its evolutionary and ecological framework. PMID:25309731

  18. Comparison of the Dictyostelium rasD and ecmA genes reveals two distinct mechanisms whereby an mRNA may become enriched in prestalk cells.

    PubMed

    Jermyn, K; Wiliams, J

    1995-04-01

    The Dictyostelium ras gene, rasD, encodes an mRNA that is more abundant in prestalk than prespore cells in the migratory slug. Its expression is inducible by extracellular cAMP but is not inducible by the prestalk and stalk cell morphogen differentiation inducing factor (DIF). We show that a rasD-lacZ fusion gene is first expressed in approximately one half of the cells in the aggregate, including some cells that also express a prespore-specific marker. The amount of rasD-lacZ fusion protein in prespore cells then diminishes as the slug is formed. Analysis of a rasD-lacZ fusion protein with an N terminal substitution that reduces protein stability within the cell provides strong confirmatory evidence that the ras gene product becomes enriched in prestalk cells by selective repression of gene expression in prespore cells. In contrast, the DIF-inducible ecmA gene is expressed only in those cells that will become prestalk cells in the migratory slug. These results show that there are two different ways in which an mRNA may become enriched in prestalk cells and support the view that DIF is the inducer of prestalk cell differentiation.

  19. Mechanical guidance through cell-cell and cell-surface contact during multicellular streaming

    NASA Astrophysics Data System (ADS)

    Wang, Chenlu; Driscoll, Meghan; Gupta, Satyandra K.; Parent, Carole; Losert, Wolfgang

    2014-03-01

    During collective cell migration, mechanical forces arise from the extracellular matrix (ECM) through cell-surface contact and from other cells through cell-cell contact. These forces regulate the motion of migrating cell groups. To determine how these mechanical interactions balance during cell migration, we measured the shape dynamics of Dictyostelium discoideum cells at the multicellular streaming stage. We found that cells can coordinate their motion by synchronizing protrusion waves that travel along their membranes when they form proper cell-cell adhesion and cell-surface adhesion. In addition, our experiments on live actin labeled cells show that intracellular actin polymerization actively responds to the change of cell-cell/surface adhesion and helps to stabilize multicellular migration streams. Our finding suggests that the coordination of motion between neighboring cells in collective migration requires a balance between cell-cell adhesion and cell-surface adhesion, and that the cell cytoskeleton plays an important role in this balance.

  20. The Gα4 G protein subunit interacts with the MAP kinase ERK2 using a D-motif that regulates developmental morphogenesis in Dictyostelium

    PubMed Central

    Nguyen, Hoai-Nghia; Hadwiger, Jeffrey A.

    2009-01-01

    G protein Gα subunits contribute to the specificity of different signal transduction pathways in Dictyostelium discoideum but Gα subunit-effector interactions have not been previously identified. The requirement of the Dictyostelium Gα4 subunit for MAP kinase (MAPK) activation and the identification of a putative MAPK docking site (D-motif) in this subunit suggested a possible interaction between the Gα4 subunit and MAPKs. In vivo association of the Gα4 subunit and ERK2 was demonstrated by pull-down and co-immunoprecipitation assays. Alteration of the D-motif reduced Gα4 subunit-ERK2 interactions but only slightly altered MAPK activation in response to folate. Expression of the Gα4 subunit with the altered D-motif in gα4− cells allowed for slug formation but not the morphogenesis associated with culmination. Expression of this mutant Gα4 subunit was sufficient to rescue chemotactic movement to folate. Alteration of the D-motif also reduced the aggregation defect associated with constitutively active Gα4 subunits. These results suggest Gα4 subunit-MAPK interactions are necessary for developmental morphogenesis but not for chemotaxis to folate. PMID:19765570

  1. Actin Foci Adhesion of D. discoideum

    NASA Astrophysics Data System (ADS)

    Flanders, Bret; Paneru, Govind

    2014-03-01

    Amoeboid migration is a fast (10 μm min-1) integrin-independent mode of migration that is important with D. discoideum, leukocytes, and breast cancer cells. It is poorly understood, but depends on the establishment of adhesive contacts to the substrate where the cell transmits traction forces. In pre-aggregative D. discoideum, a model system for learning about amoeboid migration, these adhesive contacts are discrete complexes that are known as actin-foci. They have an area of ~ 0.5 μm2 and a lifetime of ~ 20 s. This talk will present measurements of the adhesive character of actin foci that have been obtained using a submicron force transducer that was designed for this purpose. Results on the rupture stresses and lifetimes of individual acting foci under nano-newton level forces will be described in the context of a general theory for cellular adhesion. This theory depends on, essentially, three cellular properties: the membrane-medium surface tension, the number density of adhesion receptors in the membrane, and the receptor-substrate potential energy surface. Therefore, the use of the transducer to determine the surface tension will be presented, as well.

  2. Behavior of Dictyostelium amoebae is regulated primarily by the temporal dynamic of the natural cAMP wave.

    PubMed

    Wessels, D; Murray, J; Soll, D R

    1992-01-01

    The instantaneous velocity plots of Dictyostelium discoideum amoebae responding to natural waves and simulated temporal waves of cAMP with periods of 7 min are highly similar. This similarity has been used to deduce the dynamics of a natural wave crossing an amoeba, and the behavior of amoebae has been characterized during the different phases of a natural wave with a computer-assisted dynamic image analyzing system. During the first approximately 150 sec of the front of a natural wave, cells move persistently toward the aggregation center, with high instantaneous velocity and a decreased frequency of lateral pseudopod formation. During the last 30 sec of the front of the wave and the first 30 sec of the back of the wave, there is a "freeze" in cell shape and a dramatic depression in cell motility, pseudopod formation, and intracellular particle movement. During the last 180 sec of the back of the wave, there is a rebound in pseudopod formation, but it is random in direction and leads to no net cellular translocation. The data suggest that all of the behavior of a cell but orientation during the translocation phase is mediated by the temporal dynamics of the wave. The data also suggest that orientation toward the aggregation center occurs early in the front of the wave and that, once oriented, cells move in a blind fashion during the translocation phase.

  3. The C Isoform of Dictyostelium Tetraspanins Localizes to the Contractile Vacuole and Contributes to Resistance against Osmotic Stress.

    PubMed

    Albers, Tineke; Maniak, Markus; Beitz, Eric; von Bülow, Julia

    2016-01-01

    Tetraspanins (Tsps) are membrane proteins that are widely expressed in eukaryotic organisms. Only recently, Tsps have started to acquire relevance as potential new drug targets as they contribute, via protein-protein interactions, to numerous pathophysiological processes including infectious diseases and cancer. However, due to a high number of isoforms and functional redundancy, knowledge on specific functions of most Tsps is still scarce. We set out to characterize five previously annotated Tsps, TspA-E, from Dictyostelium discoideum, a model for studying proteins that have human orthologues. Using reverse transcriptase PCRs, we found mRNAs for TspA-E in the multicellular slug stage, whereas vegetative cells expressed only TspA, TspC and, to a lesser extent, TspD. We raised antibodies against TspA, TspC and TspD and detected endogenous TspA, as well as heterologously expressed TspA and TspC by Western blot. N-deglycosylation assays and mutational analyses showed glycosylation of TspA and TspC in vivo. GFP-tagged Tsps co-localized with the proton pump on the contractile vacuole network. Deletion strains of TspC and TspD exibited unaltered growth, adhesion, random motility and development. Yet, tspC- cells showed a defect in coping with hypo-osmotic stress, due to accumulation of contractile vacuoles, but heterologous expression of TspC rescued their phenotype. In conclusion, our data fill a gap in Dictyostelium research and open up the possibility that Tsps in contractile vacuoles of e.g. Trypanosoma may one day constitute a valuable drug target for treating sleeping sickness, one of the most threatening tropical diseases.

  4. The C Isoform of Dictyostelium Tetraspanins Localizes to the Contractile Vacuole and Contributes to Resistance against Osmotic Stress

    PubMed Central

    Albers, Tineke; Maniak, Markus; Beitz, Eric; von Bülow, Julia

    2016-01-01

    Tetraspanins (Tsps) are membrane proteins that are widely expressed in eukaryotic organisms. Only recently, Tsps have started to acquire relevance as potential new drug targets as they contribute, via protein-protein interactions, to numerous pathophysiological processes including infectious diseases and cancer. However, due to a high number of isoforms and functional redundancy, knowledge on specific functions of most Tsps is still scarce. We set out to characterize five previously annotated Tsps, TspA-E, from Dictyostelium discoideum, a model for studying proteins that have human orthologues. Using reverse transcriptase PCRs, we found mRNAs for TspA-E in the multicellular slug stage, whereas vegetative cells expressed only TspA, TspC and, to a lesser extent, TspD. We raised antibodies against TspA, TspC and TspD and detected endogenous TspA, as well as heterologously expressed TspA and TspC by Western blot. N-deglycosylation assays and mutational analyses showed glycosylation of TspA and TspC in vivo. GFP-tagged Tsps co-localized with the proton pump on the contractile vacuole network. Deletion strains of TspC and TspD exibited unaltered growth, adhesion, random motility and development. Yet, tspC− cells showed a defect in coping with hypo-osmotic stress, due to accumulation of contractile vacuoles, but heterologous expression of TspC rescued their phenotype. In conclusion, our data fill a gap in Dictyostelium research and open up the possibility that Tsps in contractile vacuoles of e.g. Trypanosoma may one day constitute a valuable drug target for treating sleeping sickness, one of the most threatening tropical diseases. PMID:27597994

  5. Nanotopography-induced symmetry-breaking and guidance of actin polymerization waves and cell migration

    NASA Astrophysics Data System (ADS)

    Losert, Wolfgang; Guven, Can; Sun, Xiaoyu; Fourkas, John; Carlsson, Anders; Driscoll, Meghan

    2015-03-01

    Many types of eukaryotic cells on a surfaces exhibit reaction diffusion-type waves of actin polymerization. Exposing migrating Dictyostelium discoideum cells to asymmetries at a length scale relevant to actin waves (300 nm) results in guidance of actin polymerization and of the migration of the cells themselves. Quantitative measurements of actin wave speed and direction distributions show that actin polymerization is preferentially localized to nanoridges and directed along the ridges, and that the velocity of guided actin polymerization waves decreases with decreasing ridge spacing. A stochastic growth model of actin polymerization dynamics reproduces these key observations. Supported by NSF-PoLS.

  6. Real-time visualization of intracellular hydrodynamics in single living cells.

    PubMed

    Potma, E; de Boeij, W P; van Haastert, P J; Wiersma, D A

    2001-02-13

    Intracellular water concentrations in single living cells were visualized by nonlinear coherent anti-Stokes Raman scattering (CARS) microscopy. In combination with isotopic exchange measurements, CARS microscopy allowed the real-time observation of transient intracellular hydrodynamics at a high spatial resolution. Studies of the hydrodynamics in the microorganism Dictyostelium discoideum indicated the presence of a microscopic region near the plasma membrane where the mobility of water molecules is severely restricted. Modeling the transient hydrodynamics eventuated in the determination of cell-specific cytosolic diffusion and plasma membrane permeability constants. Our experiments demonstrate that CARS microscopy offers an invaluable tool for probing single-cell water dynamics.

  7. The Dictyostelium MAPK ERK1 is phosphorylated in a secondary response to early developmental signaling.

    PubMed

    Schwebs, David J; Hadwiger, Jeffrey A

    2015-01-01

    Previous reports have suggested that the two mitogen-activated protein kinases (MAPKs) in Dictyostelium discoideum, ERK1 and ERK2, can be directly activated in response to external cAMP even though these MAPKs play different roles in the developmental life cycle. To better characterize MAPK regulation, the levels of phosphorylated MAPKs were analyzed in response to external signals. Only ERK2 was rapidly phosphorylated in response to the chemoattractants, cAMP and folate. In contrast, the phosphorylation of ERK1 occurred as a secondary or indirect response to these stimuli and this phosphorylation was enhanced by cell-cell interactions, suggesting that other external signals can activate ERK1. The phosphorylation of ERK1 or ERK2 did not require the function of the other MAPK in these responses. Folate stimulation of a chimeric population of erk1- and gα4- cells revealed that the phosphorylation of ERK1 could be mediated through an intercellular signal other than folate. Loss of ERK1 function suppressed the developmental delay and the deficiency in anterior cell localization associated with gα5- mutants suggesting that ERK1 function can be down regulated through Gα5 subunit-mediated signaling. However, no major changes in the phosphorylation of ERK1 were observed in gα5- cells suggesting that the Gα5 subunit signaling pathway does not regulate the phosphorylation of ERK1. These findings suggest that the activation of ERK1 occurs as a secondary response to chemoattractants and that other cell-cell signaling mechanisms contribute to this activation. Gα5 subunit signaling can down regulate ERK1 function to promote prestalk cell development but not through major changes to the level of phosphorylated ERK1.

  8. Dictyostelium phenylalanine hydroxylase is activated by its substrate phenylalanine.

    PubMed

    Kim, Hye-Lim; Park, Mi-Bee; Kim, Yumin; Yang, Yun Gyeong; Lee, Soo-Woong; Zhuang, Ningning; Lee, Kon Ho; Park, Young Shik

    2012-10-19

    We have studied the regulatory function of Dictyostelium discoideum Ax2 phenylalanine hydroxylase (dicPAH) via characterization of domain structures. Including the full-length protein, partial proteins truncated in regulatory, tetramerization, or both, were prepared from Escherichia coli as his-tag proteins and examined for oligomeric status and catalytic parameters for phenylalanine. The proteins were also expressed extrachromosomally in the dicPAH knockout strain to examine their in vivo compatibility. The results suggest that phenylalanine activates dicPAH, which is functional in vivo as a tetramer, although cooperativity was not observed. In addition, the results of kinetic study suggest that the regulatory domain of dicPAH may play a role different from that of the domain in mammalian PAH.

  9. Investigating the effect of emetic compounds on chemotaxis in Dictyostelium identifies a non-sentient model for bitter and hot tastant research.

    PubMed

    Robery, Steven; Mukanowa, Janina; Percie du Sert, Nathalie; Andrews, Paul L R; Williams, Robin S B

    2011-01-01

    Novel chemical entities (NCEs) may be investigated for emetic liability in a range of unpleasant experiments involving retching, vomiting or conditioned taste aversion/food avoidance in sentient animals. We have used a range of compounds with known emetic /aversive properties to examine the possibility of using the social amoeba, Dictyostelium discoideum, for research into identifying and understanding emetic liability, and hence reduce adverse animal experimentation in this area. Twenty eight emetic or taste aversive compounds were employed to investigate the acute (10 min) effect of compounds on Dictyostelium cell behaviour (shape, speed and direction of movement) in a shallow chemotaxic gradient (Dunn chamber). Compound concentrations were chosen based on those previously reported to be emetic or aversive in in vivo studies and results were recorded and quantified by automated image analysis. Dictyostelium cell motility was rapidly and strongly inhibited by four structurally distinct tastants (three bitter tasting compounds--denatonium benzoate, quinine hydrochloride, phenylthiourea, and the pungent constituent of chilli peppers--capsaicin). In addition, stomach irritants (copper chloride and copper sulphate), and a phosphodiesterase IV inhibitor also rapidly blocked movement. A concentration-dependant relationship was established for five of these compounds, showing potency of inhibition as capsaicin (IC(50) = 11.9 ± 4.0 µM) > quinine hydrochloride (IC(50) = 44.3 ± 6.8 µM) > denatonium benzoate (IC(50) = 129 ± 4 µM) > phenylthiourea (IC(50) = 366 ± 5 µM) > copper sulphate (IC(50) = 1433 ± 3 µM). In contrast, 21 compounds within the cytotoxic and receptor agonist/antagonist classes did not affect cell behaviour. Further analysis of bitter and pungent compounds showed that the effect on cell behaviour was reversible and not cytotoxic, suggesting an uncharacterised molecular mechanism of action for these compounds. These results therefore demonstrate

  10. Burkholderia bacteria infectiously induce the proto-farming symbiosis of Dictyostelium amoebae and food bacteria.

    PubMed

    DiSalvo, Susanne; Haselkorn, Tamara S; Bashir, Usman; Jimenez, Daniela; Brock, Debra A; Queller, David C; Strassmann, Joan E

    2015-09-08

    Symbiotic associations can allow an organism to acquire novel traits by accessing the genetic repertoire of its partner. In the Dictyostelium discoideum farming symbiosis, certain amoebas (termed "farmers") stably associate with bacterial partners. Farmers can suffer a reproductive cost but also gain beneficial capabilities, such as carriage of bacterial food (proto-farming) and defense against competitors. Farming status previously has been attributed to amoeba genotype, but the role of bacterial partners in its induction has not been examined. Here, we explore the role of bacterial associates in the initiation, maintenance, and phenotypic effects of the farming symbiosis. We demonstrate that two clades of farmer-associated Burkholderia isolates colonize D. discoideum nonfarmers and infectiously endow them with farmer-like characteristics, indicating that Burkholderia symbionts are a major driver of the farming phenomenon. Under food-rich conditions, Burkholderia-colonized amoebas produce fewer spores than uncolonized counterparts, with the severity of this reduction being dependent on the Burkholderia colonizer. However, the induction of food carriage by Burkholderia colonization may be considered a conditionally adaptive trait because it can confer an advantage to the amoeba host when grown in food-limiting conditions. We observed Burkholderia inside and outside colonized D. discoideum spores after fruiting body formation; this observation, together with the ability of Burkholderia to colonize new amoebas, suggests a mixed mode of symbiont transmission. These results change our understanding of the D. discoideum farming symbiosis by establishing that the bacterial partner, Burkholderia, is an important causative agent of the farming phenomenon.

  11. The Mechanosensory Ca2+ Channel as a Central Regulator of Prostate Tumor Cell Migration and Invasiveness

    DTIC Science & Technology

    2008-01-01

    aquaporin‐1 gene disruption. Nature 434, 786–792. Sachs, F., and Morris, C . E. (1998). Mechanosensitive ion channels in nonspecialized cells. Revs. Physiol...Happle,K.,Malchow,D., andSchlatterer, C . (2005). Ca2þ regulation in the absence of the iplA gene product inDictyostelium discoideum. BMC Cell Biol. 6...Chalfie M (1995) A stomatin-like protein necessary for mechano- sensation in C . elegans . Nature 378:292–295 Huber TB, Scherner B, Müller RU, Höhne M

  12. Two Dictyostelium ribosomal proteins act as RNases for specific classes of mRNAs.

    PubMed Central

    Mangiarotti, Giorgio

    2003-01-01

    Phosphorylation of ribosomal protein S6 leads to the stabilization of pre-spore specific mRNAs during development of Dictyostelium discoideum. The purification of S6 kinase has allowed the identification of protein S11 as the mRNase specific for pre-spore mRNAs. Methylation of ribosomal protein S31 leads to the destabilization of ribosomal protein mRNAs. The purification of S31 methyltransferase has allowed the identification of protein S29 as the mRNAse specific for ribosomal protein mRNAs. PMID:12392449

  13. Two Dictyostelium ribosomal proteins act as RNases for specific classes of mRNAs.

    PubMed

    Mangiarotti, Giorgio

    2003-03-01

    Phosphorylation of ribosomal protein S6 leads to the stabilization of pre-spore specific mRNAs during development of Dictyostelium discoideum. The purification of S6 kinase has allowed the identification of protein S11 as the mRNase specific for pre-spore mRNAs. Methylation of ribosomal protein S31 leads to the destabilization of ribosomal protein mRNAs. The purification of S31 methyltransferase has allowed the identification of protein S29 as the mRNAse specific for ribosomal protein mRNAs.

  14. Collective Promotion of Cell Proliferation in an Eukaryotic Suspension Culture

    NASA Astrophysics Data System (ADS)

    Franck, Carl

    2012-02-01

    We argue that the well known transition from slow to fast population growth with time in a well mixed suspension of the amoeba Dictyostelium discoideum relies on long range chemical communication, not cell contacts as we had argued earlier (Phys. Rev. E v. 77, 041905 (2008)). We show that while such a mode of communication is biochemically plausible, an explanation for the significant variation in growth we have measured is lacking. Since the transition density is low this system offers an elegantly simple example of a multicellular life process.

  15. Steroids initiate a signaling cascade that triggers rapid sporulation in Dictyostelium

    PubMed Central

    Anjard, Christophe; Su, Yongxuan; Loomis, William F.

    2009-01-01

    Summary Encapsulation of prespore cells of Dictyostelium discoideum is controlled by several intercellular signals to ensure appropriate timing during fruiting body formation. Acyl-CoA-binding protein, AcbA, is secreted by prespore cells and processed by the prestalk protease TagC to form the 34 amino acid peptide SDF-2 that triggers rapid encapsulation. AcbA is secreted when γ-aminobutyric acid (GABA) is released from prespore cells and binds to GrlE, a G protein-coupled receptor (GPCR). Analysis of SDF-2 production in mutant strains lacking Gα subunits and GPCRs, either as pure populations or when mixed with other mutant strains, uncovered the non-cell-autonomous roles of GrlA, Gα4 and Gα7. We found that Gα7 is essential for the response to GABA and is likely to be coupled to GrlE. GrlA-null and Gα4-null cells respond normally to GABA but fail to secrete it. We found that they are necessary for the response to a small hydrophobic molecule, SDF-3, which is released late in culmination. Pharmacological inhibition of steroidogenesis during development blocked the production of SDF-3. Moreover, the response to SDF-3 could be blocked by the steroid antagonist mifepristone, whereas hydrocortisone and other steroids mimicked the effects of SDF-3 when added in the nanomolar range. It appears that SDF-3 is a steroid that elicits rapid release of GABA by acting through the GPCR GrlA, coupled to G protein containing the Gα4 subunit. SDF-3 is at the head of the cascade that amplifies the signal for encapsulation to ensure the rapid, synchronous formation of spores. PMID:19176583

  16. Discovery of myosin genes by physical mapping in Dictyostelium.

    PubMed Central

    Titus, M A; Kuspa, A; Loomis, W F

    1994-01-01

    The diversity of the myosin family in a single organism, Dictyostelium discoideum, has been investigated by a strategy devised to rapidly identify and clone additional members of a gene family. An ordered array of yeast artificial chromosome clones that encompasses the Dictyostelium genome was probed at low stringency with conserved regions of the myosin motor domain to identify all possible myosin loci. The previously identified myosin loci (mchA, myoA-E) were detected by hybridization to the probes, as well as an additional seven previously unidentified loci (referred to as myoF-L). Clones corresponding to four of these additional loci (myoF, myoH-J) were obtained by using the isolated yeast artificial chromosomes as templates in a PCR employing degenerate primers specific for conserved regions of the myosin head. Sequence analysis and physical mapping of these clones confirm that these PCR products are derived from four previously unidentified myosin genes. Preliminary analysis of these sequences suggests that at least one of the genes (myoJ) encodes a member of a potentially different class of myosins. With the development of whole genome libraries for a variety of organisms, this approach can be used to rapidly explore the diversity of this and other gene families in a number of systems. PMID:7937787

  17. Evidence that the Dictyostelium Dd-STATa protein is a repressor that regulates commitment to stalk cell differentiation and is also required for efficient chemotaxis.

    PubMed

    Mohanty, S; Jermyn, K A; Early, A; Kawata, T; Aubry, L; Ceccarelli, A; Schaap, P; Williams, J G; Firtel, R A

    1999-08-01

    Dd-STATa is a structural and functional homologue of the metazoan STAT (Signal Transducer and Activator of Transcription) proteins. We show that Dd-STATa null cells exhibit several distinct developmental phenotypes. The aggregation of Dd-STATa null cells is delayed and they chemotax slowly to a cyclic AMP source, suggesting a role for Dd-STATa in these early processes. In Dd-STATa null strains, slug-like structures are formed but they have an aberrant pattern of gene expression. In such slugs, ecmB/lacZ, a marker that is normally specific for cells on the stalk cell differentiation pathway, is expressed throughout the prestalk region. Stalk cell differentiation in Dictyostelium has been proposed to be under negative control, mediated by repressor elements present in the promoters of stalk cell-specific genes. Dd-STATa binds these repressor elements in vitro and the ectopic expression of ecmB/lacZ in the null strain provides in vivo evidence that Dd-STATa is the repressor protein that regulates commitment to stalk cell differentiation. Dd-STATa null cells display aberrant behavior in a monolayer assay wherein stalk cell differentiation is induced using the stalk cell morphogen DIF. The ecmB gene, a general marker for stalk cell differentiation, is greatly overinduced by DIF in Dd-STATa null cells. Also, Dd-STATa null cells are hypersensitive to DIF for expression of ST/lacZ, a marker for the earliest stages in the differentiation of one of the stalk cell sub-types. We suggest that both these manifestations of DIF hypersensitivity in the null strain result from the balance between activation and repression of the promoter elements being tipped in favor of activation when the repressor is absent. Paradoxically, although Dd-STATa null cells are hypersensitive to the inducing effects of DIF and readily form stalk cells in monolayer assay, the Dd-STATa null cells show little or no terminal stalk cell differentiation within the slug. Dd-STATa null slugs remain

  18. Spalten, a protein containing Galpha-protein-like and PP2C domains, is essential for cell-type differentiation in Dictyostelium.

    PubMed

    Aubry, L; Firtel, R A

    1998-05-15

    We have identified a novel gene, Spalten (Spn) that is essential for Dictyostelium multicellular development. Spn encodes a protein with an amino-terminal domain that shows very high homology to Galpha-protein subunits, a highly charged inter-region, and a carboxy-terminal domain that encodes a functional PP2C. Spn is essential for development past the mound stage, being required cell autonomously for prestalk gene expression and nonautonomously for prespore cell differentiation. Mutational analysis demonstrates that the PP2C domain is the Spn effector domain and is essential for Spn function, whereas the Galpha-like domain is required for membrane targeting and regulation of Spn function. Moreover, Spn carrying mutations in the Galpha-like domain that do not affect membrane targeting but affect specificity of guanine nucleotide binding in known GTP-binding proteins are unable to fully complement the spn- phenotype, suggesting that the Galpha-like domain regulates Spn function either directly or indirectly by mediating its interactions with other proteins. Our results suggest that Spn encodes a signaling molecule with a novel Galpha-like regulatory domain.

  19. Three-dimensional in vivo analysis of Dictyostelium mounds reveals directional sorting of prestalk cells and defines a role for the myosin II regulatory light chain in prestalk cell sorting and tip protrusion.

    PubMed

    Clow, P A; Chen, T; Chisholm, R L; McNally, J G

    2000-06-01

    During cell sorting in Dictyostelium, we observed that GFP-tagged prestalk cells (ecmAO-expressing cells) moved independently and directionally to form a cluster. This is consistent with a chemotaxis model for cell sorting (and not differential adhesion) in which a long-range signal attracts many of the prestalk cells to the site of cluster formation. Surprisingly, the ecmAO prestalk cluster that we observed was initially found at a random location within the mound of this Ax3 strain, defining an intermediate sorting stage not widely reported in Dictyostelium. The cluster then moved en masse to the top of the mound to produce the classic, apical pattern of ecmAO prestalk cells. Migration of the cluster was also directional, suggesting the presence of another long-range guidance cue. Once at the mound apex, the cluster continued moving upward leading to protrusion of the mound's tip. To investigate the role of the cluster in tip protrusion, we examined ecmAO prestalk-cell sorting in a myosin II regulatory light chain (RLC) null in which tips fail to form. In RLC-null mounds, ecmAO prestalk cells formed an initial cluster that began to move to the mound apex, but then arrested as a vertical column that extended from the mound's apex to its base. Mixing experiments with wild-type cells demonstrated that the RLC-null ecmAO prestalk-cell defect is cell autonomous. These observations define a specific mechanism for myosin's function in tip formation, namely a mechanical role in the upward movement of the ecmAO prestalk cluster. The wild-type data demonstrate that cell sorting can occur in two steps, suggesting that, in this Ax3 strain, spatially and temporally distinct cues may guide prestalk cells first to an initial cluster and then later to the tip.

  20. Biochemical and structural characterizations of two Dictyostelium cellobiohydrolases from the amoebozoa kingdom reveal a high level of conservation between distant phylogenetic trees of life

    SciTech Connect

    Hobdey, Sarah E.; Knott, Brandon C.; Momeni, Majid Haddad; Taylor, II, Larry E.; Borisova, Anna S.; Podkaminer, Kara K.; VanderWall, Todd A.; Himmel, Michael E.; Decker, Stephen R.; Beckham, Gregg T.; Stahlberg, Jerry

    2016-04-01

    Glycoside hydrolase family 7 (GH7) cellobiohydrolases (CBHs) are enzymes often employed in plant cell wall degradation across eukaryotic kingdoms of life, as they provide significant hydrolytic potential in cellulose turnover. To date, many fungal GH7 CBHs have been examined, yet many questions regarding structure-activity relationships in these important natural and commercial enzymes remain. Here, we present the crystal structures and a biochemical analysis of two GH7 CBHs from social amoeba: Dictyostelium discoideum Cel7A (DdiCel7A) and Dictyostelium purpureum Cel7A (DpuCel7A). DdiCel7A and DpuCel7A natively consist of a catalytic domain and do not exhibit a carbohydrate-binding module (CBM). The structures of DdiCel7A and DpuCel7A, resolved to 2.1 Å and 2.7 Å, respectively, are homologous to those of other GH7 CBHs with an enclosed active-site tunnel. Two primary differences between the Dictyostelium CBHs and the archetypal model GH7 CBH, Trichoderma reesei Cel7A (TreCel7A), occur near the hydrolytic active site and the product-binding sites. To compare the activities of these enzymes with the activity of TreCel7A, the family 1 TreCel7A CBM and linker were added to the C terminus of each of the Dictyostelium enzymes, creating DdiCel7ACBM and DpuCel7ACBM, which were recombinantly expressed in T. reesei. DdiCel7ACBM and DpuCel7ACBM hydrolyzed Avicel, pretreated corn stover, and phosphoric acid-swollen cellulose as efficiently as TreCel7A when hydrolysis was compared at their temperature optima. The Ki of cellobiose was significantly higher for DdiCel7ACBM and DpuCel7ACBM than for TreCel7A: 205, 130, and 29 μM, respectively. Finally, taken together, the present study highlights the remarkable degree of conservation of the activity of these key natural and industrial enzymes across quite distant phylogenetic trees of life.

  1. Biochemical and structural characterizations of two Dictyostelium cellobiohydrolases from the amoebozoa kingdom reveal a high level of conservation between distant phylogenetic trees of life

    DOE PAGES

    Hobdey, Sarah E.; Knott, Brandon C.; Momeni, Majid Haddad; ...

    2016-04-01

    Glycoside hydrolase family 7 (GH7) cellobiohydrolases (CBHs) are enzymes often employed in plant cell wall degradation across eukaryotic kingdoms of life, as they provide significant hydrolytic potential in cellulose turnover. To date, many fungal GH7 CBHs have been examined, yet many questions regarding structure-activity relationships in these important natural and commercial enzymes remain. Here, we present the crystal structures and a biochemical analysis of two GH7 CBHs from social amoeba: Dictyostelium discoideum Cel7A (DdiCel7A) and Dictyostelium purpureum Cel7A (DpuCel7A). DdiCel7A and DpuCel7A natively consist of a catalytic domain and do not exhibit a carbohydrate-binding module (CBM). The structures of DdiCel7Amore » and DpuCel7A, resolved to 2.1 Å and 2.7 Å, respectively, are homologous to those of other GH7 CBHs with an enclosed active-site tunnel. Two primary differences between the Dictyostelium CBHs and the archetypal model GH7 CBH, Trichoderma reesei Cel7A (TreCel7A), occur near the hydrolytic active site and the product-binding sites. To compare the activities of these enzymes with the activity of TreCel7A, the family 1 TreCel7A CBM and linker were added to the C terminus of each of the Dictyostelium enzymes, creating DdiCel7ACBM and DpuCel7ACBM, which were recombinantly expressed in T. reesei. DdiCel7ACBM and DpuCel7ACBM hydrolyzed Avicel, pretreated corn stover, and phosphoric acid-swollen cellulose as efficiently as TreCel7A when hydrolysis was compared at their temperature optima. The Ki of cellobiose was significantly higher for DdiCel7ACBM and DpuCel7ACBM than for TreCel7A: 205, 130, and 29 μM, respectively. Finally, taken together, the present study highlights the remarkable degree of conservation of the activity of these key natural and industrial enzymes across quite distant phylogenetic trees of life.« less

  2. Evidence that the RdeA protein is a component of a multistep phosphorelay modulating rate of development in Dictyostelium.

    PubMed Central

    Chang, W T; Thomason, P A; Gross, J D; Neweil, P C

    1998-01-01

    We have isolated an insertional mutant of Dictyostelium discoideum that aggregated rapidly and formed spores and stalk cells within 14 h of development instead of the normal 24 h. We have shown by parasexual genetics that the insertion is in the rdeA locus and have cloned the gene. It encodes a predicted 28 kDa protein (RdeA) that is enriched in charged residues and is very hydrophilic. Constructs with the DNA for the c-Myc epitope or for the green fluorescent protein indicate that RdeA is not compartmentalized. RdeA displays homology around a histidine residue at amino acid 65 with members of the H2 module family of phosphotransferases that participate in multistep phosphoryl relays. Replacement of this histidine rendered the protein inactive. The mutant is complemented by transformation with the Ypd1 gene of Saccharomyces cerevisiae, itself an H2 module protein. We propose that RdeA is part of a multistep phosphorelay system that modulates the rate of development. PMID:9582274

  3. Ponticulin is the major high affinity link between the plasma membrane and the cortical actin network in Dictyostelium

    PubMed Central

    1994-01-01

    Interactions between the plasma membrane and underlying actin-based cortex have been implicated in membrane organization and stability, the control of cell shape, and various motile processes. To ascertain the function of high affinity actin-membrane associations, we have disrupted by homologous recombination the gene encoding ponticulin, the major high affinity actin-membrane link in Dictyostelium discoideum amoebae. Cells lacking detectable amounts of ponticulin message and protein also are deficient in high affinity actin-membrane binding by several criteria. First, only 10-13% as much endogenous actin cosediments through sucrose and crude plasma membranes from ponticulin- minus cells, as compared with membranes from the parental strain. Second, purified plasma membranes exhibit little or no binding or nucleation of exogenous actin in vitro. Finally, only 10-30% as much endogenous actin partitions with plasma membranes from ponticulin-minus cells after these cells are mechanically unroofed with polylysine- coated coverslips. The loss of the cell's major actin-binding membrane protein appears to be surprisingly benign under laboratory conditions. Ponticulin-minus cells grow normally in axenic culture and pinocytose FITC-dextran at the same rate as do parental cells. The rate of phagocytosis of particles by ponticulin-minus cells in growth media also is unaffected. By contrast, after initiation of development, cells lacking ponticulin aggregate faster than the parental cells. Subsequent morphogenesis proceeds asynchronously, but viable spores can form. These results indicate that ponticulin is not required for cellular translocation, but apparently plays a role in cell patterning during development. PMID:8089176

  4. The regulation of chemotaxis and chemokinesis in Dictyostelium amoebae by temporal signals and spatial gradients of cyclic AMP.

    PubMed

    Vicker, M G

    1994-02-01

    The tactic and kinetic locomotion of Dictyostelium discoideum amoebae were examined in cyclic AMP (cAMP) spatial gradient and temporal signal fields. The distributions of migrating cells were examined within 150 microns-thick micropore filters after incubation with different cAMP concentrations, [cAMP], applied in three ways across the fields: as positively or negatively developing gradients, generated either by increasing or decreasing the [cAMP] on one side of the filter, respectively, or as static, linear gradients after negative development. Chemotaxis was only induced by oriented, temporally increasing [cAMP]. Pulses propagated by molecular diffusion or mechanical flow were equally effective. Negatively developing cAMP gradients had no initial effect on cell accumulation. However, if the subsequent static spatial gradient was maintained by an infusion system, some gradients also induced cell accumulation, whose degree and direction depended on the gradient [cAMP]. The basis of this new effect was examined by tracking individual cells by computer-assisted videomicroscopy during locomotion in different [cAMP]. Cells produced a triphasic [cAMP]-dependent response, with optimal cell motility induced by 10-30 nM. The results demonstrate that cell accumulation either up-field or down-field in spatial gradients is governed by the field locations of the attractant concentrations that induce the relative locomotory maxima and minima in the gradient field. Cells perceive the ambient [cAMP], but cannot read the spatial gradient orientation in static or yet steeper regions of developing gradients. Accumulation in static spatial gradients is a function of klino- and orthokinesis, but chemotaxis requires an oriented cAMP pulse or impulse.(ABSTRACT TRUNCATED AT 250 WORDS)

  5. Cell Shape Dynamics: From Waves to Migration

    NASA Astrophysics Data System (ADS)

    Driscoll, Meghan; McCann, Colin; Kopace, Rael; Homan, Tess; Fourkas, John; Parent, Carole; Losert, Wolfgang

    2011-03-01

    We analyzed the dynamic shape of migrating Dictyostelium discoideum cells. We found that regions of high boundary curvature propagate from the front to the back of cells in an organized fashion. These waves of high curvature are stabilized by surface contact, and so, at the sides of cells, are stationary relative to the surface. The initiation of curvature waves, though, which usually occurs at the front of cells, is associated with protrusive motion. The protrusion location shifts rapidly in a ballistic manner at speeds nearly double that of cellular migration. To examine curvature waves in the absence of surface contact, we guided cells to extend over the edge of micro-cliffs. The curvature wave speed of cells extended over a cliff was triple the wave speed of cells migrating on a surface, which is consistent with the higher wave speeds observed near the non-adherent leading edge of cells.

  6. Functional overlap of the dictyostelium RasG, RasD and RasB proteins.

    PubMed

    Khosla, M; Spiegelman, G B; Insall, R; Weeks, G

    2000-04-01

    Disruption of the rasG gene in Dictyostelium discoideum results in several distinct phenotypes: a defect in cytokinesis, reduced motility and reduced growth. Reintroduction of the rasG gene restores all of the properties of the rasG(-) cells to those of the wild type. To determine whether the defects are due to impaired interactions with a single or multiple downstream effectors, we tested the ability of the highly related but non identical Dictyostelium ras genes, rasD and rasB, to rescue the defects. Introduction of the rasD gene under the control of the rasG promoter into rasG null (rasG(-)) cells corrected all phenotypes except the motility defect, suggesting that motility is regulated by a RasG mediated pathway that is different to those regulating growth or cytokinesis. Western blot analysis of RasD protein levels revealed that vegetative rasG(- )cells contained considerably more protein than the parental AX-3 cells, suggesting that RasD protein levels are negatively regulated in vegetative cells by RasG. The level of RasD was enhanced when the rasD gene was introduced under the control of the rasG promoter, and this increase in protein is presumably responsible for the reversal of the growth and cytokinesis defects of the rasG(- )cells. Thus, RasD protein levels are controlled by the level of RasG, but not by the level of RasD. Introduction of the rasB gene under the control of the rasG promoter into rasG(-) cells produced a complex phenotype. The transformants were extremely small and mononucleate and exhibited enhanced motility. However, the growth of these cells was considerably slower than the growth of the rasG(-) cells, suggesting the possibility that high levels of RasB inhibit an essential process. This was confirmed by expressing rasB in wild-type cells; the resulting transformants exhibited severely impaired growth. When RasB protein levels were determined by western blot analysis, it was found that levels were higher in the rasG(- )cells than they

  7. Cell-cell interactions stabilize emerging collective migration modes

    NASA Astrophysics Data System (ADS)

    Parker, Joshua; Guven, Can; Wang, Chenlu; Ott, Ed; Losert, Wolfgang

    2014-03-01

    We propose a coarse-grained mechanistic model for simulating the dynamics of the biological model organism Dictyostelium discoideum, incorporating gradient sensing, random motility via actin protrusions, persistent random motion and signal relay. We demonstrate that our simple cell model does result in the macroscopic group migration patterns seen in no-flow gradient chambers, namely a transition from individual motion to multi-cell ``streaming'' to aggregation as the external signal is decreased. We also find that cell-cell adhesion further stabilizes the contact network independent of chemical signaling, suggesting no indirect feedback between mechanical forces and gradient sensing. We discuss further modifications to the model and as well as further applications to quantifying dynamics using spatio-temporal contact networks. Co-first author

  8. Analysis of the promoter of the cudA gene reveals novel mechanisms of Dictyostelium cell type differentiation.

    PubMed

    Fukuzawa, M; Williams, J G

    2000-06-01

    The cudA gene encodes a nuclear protein that is essential for normal multicellular development. At the slug stage cudA is expressed in the prespore cells and in a sub-region of the prestalk zone. We show that cap site distal promoter sequences direct cudA expression in prespore cells, while proximal sequences direct expression in the prestalk sub-region. The promoter domain that directs prespore-specific transcription consists of a positively acting region, that has the potential to direct expression in all cells within the slug, and a negatively acting region that prevents expression in the prestalk cells. Dd-STATa is the STAT protein that regulates commitment to stalk cell gene expression, where it is known to function as a transcriptional repressor. We show that Dd-STATa binds in vitro to the positively acting part of the prespore domain of the cudA promoter. However, Dd-STATa cannot be utilised for this purpose in vivo, because analysis of a Dd-STATa null mutant strain shows that Dd-STATa is not necessary for cudA transcription in prespore cells. In contrast, the part of the cudA promoter that directs prestalk-specific expression contains a binding site for Dd-STATa that is essential for its biological activity. Dd-STATa appears therefore to serve as a direct activator of cudA transcription in prestalk cells, while a protein with a DNA binding specificity highly related to that of Dd-STATa is utilised to activate cudA transcription in prespore cells.

  9. 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.

    PubMed

    Shimada, Nao; Maruo, Toshinari; Maeda, Mineko; Urushihara, Hideko; Kawata, Takefumi

    2005-02-01

    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.

  10. Extracellular and intracellular factors regulating the migration direction of a chemotactic cell in traveling-wave chemotaxis

    NASA Astrophysics Data System (ADS)

    Ishiwata, R.; Iwasa, M.

    2015-04-01

    This report presents a simple model that describes the motion of a single Dictyostelium discoideum cell exposed to a traveling wave of cyclic adenosine monophosphate (cAMP). The model incorporates two types of responses to stimulation by cAMP: the changes in the polarity and motility of the cell. The periodic change in motility is assumed to be induced by periodic cAMP stimulation on the basis of previous experimental studies. Consequently, the net migration of the cell occurs in a particular direction with respect to wave propagation, which explains the migration of D. discoideum cells in aggregation. The wave period and the difference between the two response times are important parameters that determine the direction of migration. The theoretical prediction compared with experiments presented in another study. The transition from the single-cell state of the population of D. discoideum cells to the aggregation state is understood to be a specific example of spontaneous breakage of symmetry in biology.

  11. Dynamic contact guidance of migrating cells

    NASA Astrophysics Data System (ADS)

    Losert, Wolfgang; Sun, Xiaoyu; Guven, Can; Driscoll, Meghan; Fourkas, John

    2014-03-01

    We investigate the effects of nanotopographical surfaces on the cell migration and cell shape dynamics of the amoeba Dictyostelium discoideum. Amoeboid motion exhibits significant contact guidance along surfaces with nanoscale ridges or grooves. We show quantitatively that nanoridges spaced 1.5 μm apart exhibit the greatest contact guidance efficiency. Using principal component analysis, we characterize the dynamics of the cell shape modulated by the coupling between the cell membrane and ridges. We show that motion parallel to the ridges is enhanced, while the turning, at the largest spatial scales, is suppressed. Since protrusion dynamics are principally governed by actin dynamics, we imaged the actin polymerization of cells on ridges. We found that actin polymerization occurs preferentially along nanoridges in a ``monorail'' like fashion. The ridges then provide us with a tool to study actin dynamics in an effectively reduced dimensional system.

  12. DNA double-strand break repair pathway choice in Dictyostelium.

    PubMed

    Hsu, Duen-Wei; Kiely, Rhian; Couto, C Anne-Marie; Wang, Hong-Yu; Hudson, Jessica J R; Borer, Christine; Pears, Catherine J; Lakin, Nicholas D

    2011-05-15

    DNA double-strand breaks (DSBs) can be repaired by homologous recombination (HR) or non-homologous end joining (NHEJ). The mechanisms that govern whether a DSB is repaired by NHEJ or HR remain unclear. Here, we characterise DSB repair in the amoeba Dictyostelium. HR is the principal pathway responsible for resistance to DSBs during vegetative cell growth, a stage of the life cycle when cells are predominantly in G2. However, we illustrate that restriction-enzyme-mediated integration of DNA into the Dictyostelium genome is possible during this stage of the life cycle and that this is mediated by an active NHEJ pathway. We illustrate that Dclre1, a protein with similarity to the vertebrate NHEJ factor Artemis, is required for NHEJ independently of DNA termini complexity. Although vegetative dclre1(-) cells are not radiosensitive, they exhibit delayed DSB repair, further supporting a role for NHEJ during this stage of the life cycle. By contrast, cells lacking the Ku80 component of the Ku heterodimer that binds DNA ends to facilitate NHEJ exhibit no such defect and deletion of ku80 suppresses the DSB repair defect of dclre1(-) cells through increasing HR efficiency. These data illustrate a functional NHEJ pathway in vegetative Dictyostelium and the importance of Ku in regulating DSB repair choice during this phase of the life cycle.

  13. Large scale spontaneous synchronization of cell cycles in amoebae

    NASA Astrophysics Data System (ADS)

    Segota, Igor; Boulet, Laurent; Franck, Carl

    2014-03-01

    Unicellular eukaryotic amoebae Dictyostelium discoideum are generally believed to grow in their vegetative state as single cells until starvation, when their collective aspect emerges and they differentiate to form a multicellular slime mold. While major efforts continue to be aimed at their starvation-induced social aspect, our understanding of population dynamics and cell cycle in the vegetative growth phase has remained incomplete. We show that substrate-growtn cell populations spontaneously synchronize their cell cycles within several hours. These collective population-wide cell cycle oscillations span millimeter length scales and can be completely suppressed by washing away putative cell-secreted signals, implying signaling by means of a diffusible growth factor or mitogen. These observations give strong evidence for collective proliferation behavior in the vegetative state and provide opportunities for synchronization theories beyond classic Kuramoto models.

  14. Shell tension forces propel Dictyostelium slugs forward

    NASA Astrophysics Data System (ADS)

    Rieu, Jean-Paul; Delanoë-Ayari, Hélène

    2012-12-01

    The Dictyostelium slug is an excellent model system for studying collective movements, as it is comprised of about 105 cells all moving together in the same direction. It still remains unclear how this movement occurs and what the physical mechanisms behind it are. By applying our recently developed 3D traction force microscopy, we propose a simple explanation for slug propulsion. Most of the forces are exerted by the sheath surrounding the slug. This secreted shell is under a rather uniform tension (around 50 mN m-1) and will give rise to a tissue under pressure. Finally, we propose that this pressure will naturally push the slug tip forwards if a gradient of shell mechanical properties takes place in the very anterior part of the raised tip.

  15. Neurofibromin controls macropinocytosis and phagocytosis in Dictyostelium.

    PubMed

    Bloomfield, Gareth; Traynor, David; Sander, Sophia P; Veltman, Douwe M; Pachebat, Justin A; Kay, Robert R

    2015-03-27

    Cells use phagocytosis and macropinocytosis to internalise bulk material, which in phagotrophic organisms supplies the nutrients necessary for growth. Wildtype Dictyostelium amoebae feed on bacteria, but for decades laboratory work has relied on axenic mutants that can also grow on liquid media. We used forward genetics to identify the causative gene underlying this phenotype. This gene encodes the RasGAP Neurofibromin (NF1). Loss of NF1 enables axenic growth by increasing fluid uptake. Mutants form outsized macropinosomes which are promoted by greater Ras and PI3K activity at sites of endocytosis. Relatedly, NF1 mutants can ingest larger-than-normal particles using phagocytosis. An NF1 reporter is recruited to nascent macropinosomes, suggesting that NF1 limits their size by locally inhibiting Ras signalling. Our results link NF1 with macropinocytosis and phagocytosis for the first time, and we propose that NF1 evolved in early phagotrophs to spatially modulate Ras activity, thereby constraining and shaping their feeding structures.

  16. Selection for Spiral Waves in the Social Amoebae Dictyostelium

    NASA Astrophysics Data System (ADS)

    Palsson, Eirikur; Lee, Kyoung J.; Goldstein, Raymond E.; Franke, Jakob; Kessin, Richard H.; Cox, Edward C.

    1997-12-01

    Starving Dictyostelium amoebae emit pulses of the chemoattractant cAMP that are relayed from cell to cell as circular and spiral waves. We have recently modeled spiral wave formation in Dictyostelium. Our model suggests that a secreted protein inhibitor of an extracellular cAMP phosphodiesterase selects for spirals. Herein we test the essential features of this prediction by comparing wave propagation in wild type and inhibitor mutants. We find that mutants rarely form spirals. The territory size of mutant strains is approximately 50 times smaller than wild type, and the mature fruiting bodies are smaller but otherwise normal. These results identify a mechanism for selecting one wave symmetry over another in an excitable system and suggest that the phosphodiesterase inhibitor may be under selection because it helps regulate territory size.

  17. Functional analysis of the catalytic subunit of Dictyostelium PKA in vivo.

    PubMed

    Dammann, H; Traincard, F; Anjard, C; van Bemmelen, M X; Reymond, C; Véron, M

    1998-03-01

    The catalytic subunit of the cAMP-dependent protein kinase (PKA) from Dictyostelium discoideum contains several domains, including an unusually long N-terminal extension preceding a highly conserved catalytic core. We transformed the aggregationless PkaC-null strain with several deletion constructs of both domains. Strains transformed with genes expressing catalytically-inactive polypeptides could not rescue development. Cotransformation with constructs encoding the N-terminal extension and the catalytic core, both unable to rescue development by themselves, yielded transformants able to proceed to late development. A 27-amino acid long hydrophobic region, immediately upstream of the catalytic core, was found indispensable for PKA function. A putative role of this sequence in the acquisition of the active conformation of the protein is discussed.

  18. The effects of transcription on the nucleosome structure of four Dictyostelium genes.

    PubMed Central

    Pavlovic, J; Banz, E; Parish, R W

    1989-01-01

    Micrococcal nuclease digestion of Dictyostelium discoideum nuclei from various developmental stages was used to investigate transcription-related changes in the chromatin structure of the coding region of four genes. Gene activity was determined by Northern blotting and nuclear run on experiments. During strong transcription of the developmentally regulated cysteine proteinase I gene, a smear superimposed on a nucleosomal ladder was observed, indicating perturbation of nucleosomal structure was occurring. However, two other developmentally regulated genes, discoidin I and pSC253, showed only slight nucleosome disruption during high levels of transcription. The chromatin structure of a fourth gene (pCZ22) was disrupted throughout development, even at those stages where transcription was greatly reduced. We suggest that although nucleosome structure can be transiently perturbed by the passage of the transcription complex in vivo, the degree of perturbation and the speed with which nucleosomes reassemble is also influenced by the DNA sequence. Images PMID:2704621

  19. Establishing direction during chemotaxis in eukaryotic cells.

    PubMed Central

    Rappel, Wouter-Jan; Thomas, Peter J; Levine, Herbert; Loomis, William F

    2002-01-01

    Several recent studies have demonstrated that eukaryotic cells, including amoeboid cells of Dictyostelium discoideum and neutrophils, respond to chemoattractants by translocation of PH-domain proteins to the cell membrane, where these proteins participate in the modulation of the cytoskeleton and relay of the signal. When the chemoattractant is released from a pipette, the localization is found predominantly on the proximal side of the cell. The recruitment of PH-domain proteins, particularly for Dictyostelium cells, occurs very rapidly (<2 s). Thus, the mechanism responsible for the first step in the directional sensing process of a cell must be able to establish an asymmetry on the same time scale. Here, we propose a simple mechanism in which a second messenger, generated by local activation of the membrane, diffuses through the interior of the cell, suppresses the activation of the back of the cell, and converts the temporal gradient into an initial cellular asymmetry. Numerical simulations show that such a mechanism is plausible. Available evidence suggests that the internal inhibitor may be cGMP, which accumulates within less than a second following treatment of cells with external cAMP. PMID:12202361

  20. Memory improves precision of cell sensing in fluctuating environments

    NASA Astrophysics Data System (ADS)

    Aquino, Gerardo; Tweedy, Luke; Heinrich, Doris; Endres, Robert G.

    2014-07-01

    Biological cells are often found to sense their chemical environment near the single-molecule detection limit. Surprisingly, this precision is higher than simple estimates of the fundamental physical limit, hinting towards active sensing strategies. In this work, we analyse the effect of cell memory, e.g. from slow biochemical processes, on the precision of sensing by cell-surface receptors. We derive analytical formulas, which show that memory significantly improves sensing in weakly fluctuating environments. However, surprisingly when memory is adjusted dynamically, the precision is always improved, even in strongly fluctuating environments. In support of this prediction we quantify the directional biases in chemotactic Dictyostelium discoideum cells in a flow chamber with alternating chemical gradients. The strong similarities between cell sensing and control engineering suggest universal problem-solving strategies of living matter.

  1. Memory improves precision of cell sensing in fluctuating environments

    PubMed Central

    Aquino, Gerardo; Tweedy, Luke; Heinrich, Doris; Endres, Robert G.

    2014-01-01

    Biological cells are often found to sense their chemical environment near the single-molecule detection limit. Surprisingly, this precision is higher than simple estimates of the fundamental physical limit, hinting towards active sensing strategies. In this work, we analyse the effect of cell memory, e.g. from slow biochemical processes, on the precision of sensing by cell-surface receptors. We derive analytical formulas, which show that memory significantly improves sensing in weakly fluctuating environments. However, surprisingly when memory is adjusted dynamically, the precision is always improved, even in strongly fluctuating environments. In support of this prediction we quantify the directional biases in chemotactic Dictyostelium discoideum cells in a flow chamber with alternating chemical gradients. The strong similarities between cell sensing and control engineering suggest universal problem-solving strategies of living matter. PMID:25023459

  2. Cell Size Clues for the Allee Effect in Vegetative Amoeba Suspension Culture

    NASA Astrophysics Data System (ADS)

    Franck, Carl; Rappazzo, Brendan; Wang, Xiaoning; Segota, Igor

    That cells proliferate at higher rates with increasing density helps us appreciate and understand the development of multicellular behavior through the study of dilute cell systems. However, arduous cell counting with a microscope reveals that in the model eukaryote, Dictyostelium discoideum this transition is difficult to ascertain and thereby further explore despite our earlier progress (Phys. Rev. E 77, 041905, (2008)). Here we report preliminary evidence that the slow proliferation phase is well characterized by reduced cell size compared to the wide distribution of cell sizes in the familiar exponential proliferation phase of moderate densities. This observation is enabled by a new system for characterizing cells in stirred suspension cultures. Our technique relies on quickly acquiring magnitude distributions of detected flashes of laser light scattered in situ by cell targets.

  3. MAPKs in development: insights from Dictyostelium signaling pathways

    PubMed Central

    Hadwiger, Jeffrey A.; Nguyen, Hoai-Nghia

    2011-01-01

    Mitogen activated protein kinases (MAPKs) play important roles in the development of eukaryotic organisms through the regulation of signal transduction pathways stimulated by external signals. MAPK signaling pathways have been associated with the regulation of cell growth, differentiation, and chemotaxis, indicating MAPKs contribute to a diverse set of developmental processes. In most eukaryotes, the diversity of external signals is likely to far exceed the diversity of MAPKs, suggesting that multiple signaling pathways might share MAPKs. Do different signaling pathways converge before MAPK function or can MAPKs maintain signaling specificity through interactions with specific proteins? The genetic and biochemical analysis of MAPK pathways in simple eukaryotes such as Dictyostelium offers opportunities to investigate functional specificity of MAPKs in G protein-mediated signal transduction pathways. This review considers the regulation and specificity of MAPK function in pathways that control Dictyostelium growth and development. PMID:21666837

  4. Identification of Dictyostelium G alpha genes expressed during multicellular development.

    PubMed Central

    Hadwiger, J A; Wilkie, T M; Strathmann, M; Firtel, R A

    1991-01-01

    Guanine nucleotide-binding protein (G protein)-mediated signal transduction constitutes a common mechanism by which cells receive and respond to a diverse set of environmental signals. Many of the signals involved in the developmental life cycle of the slime mold Dictyostelium have been postulated to be transduced by such pathways and, in some cases, these pathways have been demonstrated to be dependent on specific G proteins. Using the polymerase chain reaction, we have identified two additional Dictyostelium G alpha genes, G alpha 4 and G alpha 5, that are developmentally regulated. Transcripts from both of these genes are primarily expressed during the multicellular stages of development, suggesting possible roles in cell differentiation or morphogenesis. The entire G alpha 4 gene was sequenced and found to encode a protein consisting of 345 amino acids. The G alpha 4 subunit is homologous to other previously identified G alpha subunits, including the Dictyostelium G alpha 1 (43% identity) and G alpha 2 (41% identity) subunits. However, the G alpha 4 subunit contains some unusual sequence divergences in residues highly conserved among most eukaryotic G alpha subunits, suggesting that G alpha 4 may be a member of another class of G alpha subunits. Images PMID:1910174

  5. Multiple actin-based motor genes in Dictyostelium.

    PubMed Central

    Titus, M A; Warrick, H M; Spudich, J A

    1989-01-01

    Dictyostelium cells, devoid of conventional myosin, display a variety of motile activities, consistent with the presence of other molecular motors. The Dictyostelium genome was probed at low stringency with a gene fragment containing the conserved conventional myosin head domain sequences to identify other actin-based motors that may play a role in the observed motility of these mutant cells. One gene (abmA) has been characterized and encodes a polypeptide of approximately 135 kDa with a head region homologous to other myosin head sequences and a tail region that is not predicted to form either an alpha-helical structure of coiled-coil interactions. Comparisons of the amino acid sequences of the tail regions of abmA, Dictyostelium myosin I, and Acanthamoeba myosins IB and IL reveal an area of sequence similarity in the amino terminal half of the tail that may be a membrane-binding domain. The abmA gene, however, does not contain an unusual Gly, Pro, Ala stretch typical of many of the previously described myosin Is. Two additional genes (abmB and abmC) were identified using this approach and also found to contain sequences that encode proteins with typical conserved myosin head sequences. The abm genes may be part of a large family of actin-based motors that play various roles in diverse aspects of cellular motility. Images PMID:2519618

  6. Direct measurement of cell detachment force on single cells using a new electromechanical method.

    PubMed

    Francis, G W; Fisher, L R; Gamble, R A; Gingell, D

    1987-05-01

    We describe a new device in which an accurately measured force is applied to individual adherent cells while the topography of the adhesion zone is simultaneously monitored. The force is applied via a flexible glass micropipette, attached by suction to the cell under study, and is calculated directly from the measured pipette deflection. Regions of close contact in the adhesion zone are observed using interference reflection microscopy. We have used the device to measure the force required to detach human red blood cells from hydrophobic and hydrophilic glass surfaces, and to detach Dictyostelium discoideum amoebae from a hydrophobic glass surface. The measured forces per unit length of contact perimeter are within an order of magnitude of the tensions required for membrane rupture.

  7. Perturbing Streaming in Dictyostelium discoidium Aggregation

    NASA Astrophysics Data System (ADS)

    Rericha, Erin; Garcia, Gene; Parent, Carole; Losert, Wolfgang

    2009-03-01

    The ability of cells to move towards environmental cues is a critical process allowing the destruction of intruders by the immune system, the formation of the vascular system and the whole scale remodeling of tissues during embryo development. We examine the initial transition from single cell to group migration in the social amoeba Dictyostelium discoidium. Upon starvation, D. discoidium cells enter into a developmental program that triggers solitary cells to aggregate into a multicellular structure. The aggregation is mediated by the small molecule, cyclic-AMP, that cells sense, synthesize, secrete and migrate towards often in a head-to-tail fashion called a stream. Using experiment and numerical simulation, we study the sensitivity of streams to perturbations in the cyclic-AMP concentration field. We find the stability of the streams requires cells to shape the cyclic-AMP field through localized secretion and degradation. In addition, we find the streaming phenotype is sensitive to changes in the substrate properties, with slicker surfaces leading to longer more branched streams that yield large initial aggregates.

  8. Spontaneous emergence of large-scale cell cycle synchronization in amoeba colonies

    NASA Astrophysics Data System (ADS)

    Segota, Igor; Boulet, Laurent; Franck, David; Franck, Carl

    2014-06-01

    Unicellular eukaryotic amoebae Dictyostelium discoideum are generally believed to grow in their vegetative state as single cells until starvation, when their collective aspect emerges and they differentiate to form a multicellular slime mold. While major efforts continue to be aimed at their starvation-induced social aspect, our understanding of population dynamics and cell cycle in the vegetative growth phase has remained incomplete. Here we show that cell populations grown on a substrate spontaneously synchronize their cell cycles within several hours. These collective population-wide cell cycle oscillations span millimeter length scales and can be completely suppressed by washing away putative cell-secreted signals, implying signaling by means of a diffusible growth factor or mitogen. These observations give strong evidence for collective proliferation behavior in the vegetative state.

  9. Directional sensing and streaming in Dictyostelium aggregation

    NASA Astrophysics Data System (ADS)

    Almeida, Sofia; Dilão, Rui

    2016-05-01

    We merge the Kessler-Levine simple discrete model for Dictyostelium cyclic adenosine monophosphate (cAMP) production and diffusion with the Dilão-Hauser directional sensing aggregation mechanism. The resulting compound model describes all the known transient patterns that emerge during Dictyostelium aggregation, which include the spontaneous formation of cAMP self-sustained target and spiral waves and streaming. We show that the streaming patterns depend on the speed of the amoebae, on the relaxation time for the production of cAMP, on the cAMP degradation rate, and on directional sensing. Moreover, we show that different signaling centers emerge during Dictyostelium aggregation.

  10. Gene discovery by chemical mutagenesis and whole-genome sequencing in Dictyostelium

    PubMed Central

    Li, Cheng-Lin Frank; Santhanam, Balaji; Webb, Amanda Nicole; Zupan, Blaž

    2016-01-01

    Whole-genome sequencing is a useful approach for identification of chemical-induced lesions, but previous applications involved tedious genetic mapping to pinpoint the causative mutations. We propose that saturation mutagenesis under low mutagenic loads, followed by whole-genome sequencing, should allow direct implication of genes by identifying multiple independent alleles of each relevant gene. We tested the hypothesis by performing three genetic screens with chemical mutagenesis in the social soil amoeba Dictyostelium discoideum. Through genome sequencing, we successfully identified mutant genes with multiple alleles in near-saturation screens, including resistance to intense illumination and strong suppressors of defects in an allorecognition pathway. We tested the causality of the mutations by comparison to published data and by direct complementation tests, finding both dominant and recessive causative mutations. Therefore, our strategy provides a cost- and time-efficient approach to gene discovery by integrating chemical mutagenesis and whole-genome sequencing. The method should be applicable to many microbial systems, and it is expected to revolutionize the field of functional genomics in Dictyostelium by greatly expanding the mutation spectrum relative to other common mutagenesis methods. PMID:27307293

  11. Regulation of a LATS-homolog by Ras GTPases is important for the control of cell division

    PubMed Central

    2014-01-01

    Background Nuclear Dbf-related/large tumor suppressor (NDR/LATS) kinases have been shown recently to control pathways that regulate mitotic exit, cytokinesis, cell growth, morphological changes and apoptosis. LATS kinases are core components of the Hippo signaling cascade and important tumor suppressors controlling cell proliferation and organ size in flies and mammals, and homologs are also present in yeast and Dictyostelium discoideum. Ras proto-oncogens regulate many biological functions, including differentiation, proliferation and apoptosis. Dysfunctions of LATS kinases or Ras GTPases have been implicated in the development of a variety of cancers in humans. Results In this study we used the model organism Dictyostelium discoideum to analyze the functions of NdrC, a homolog of the mammalian LATS2 protein, and present a novel regulatory mechanism for this kinase. Deletion of the ndrC gene caused impaired cell division and loss of centrosome integrity. A yeast two-hybrid analysis, using activated Ras proteins as bait, revealed NdrC as an interactor and identified its Ras-binding domain. Further in vitro pull-down assays showed that NdrC binds RasG and RasB, and to a lesser extent RasC and Rap1. In cells lacking NdrC, the levels of activated RasB and RasG are up-regulated, suggesting a functional connection between RasB, RasG, and NdrC. Conclusions Dictyostelium discoideum NdrC is a LATS2-homologous kinase that is important for the regulation of cell division. NdrC contains a Ras-binding domain and interacts preferentially with RasB and RasG. Changed levels of both, RasB or RasG, have been shown previously to interfere with cell division. Since a defect in cell division is exhibited by NdrC-null cells, RasG-null cells, and cells overexpressing activated RasB, we propose a model for the regulation of cytokinesis by NdrC that involves the antagonistic control by RasB and RasG. PMID:24986648

  12. Analysis of Nonlinear Problems in Hydrodynamics and Reaction-Diffusion.

    DTIC Science & Technology

    1986-10-01

    evidence concerning chemotactic response in aggregating Dictyostelium discoideum." Journal of Cell Science 25, 191-204 (1977). L. A. Segel, I. Chet, and Y...34Unified mechanism for relay and oscillation of cyclic AMP in Dictyostelium discoideum." Proceedings National Academy of Sciences (U.S.A.) 4, 1543-1547 (1977...190 (1978). 4 v -~~~~. 17 -77 . - -- - - U. Parnas and L. A. Segel, "A computer simulation of pulsatile aggregation in Dictyostelium discoideum

  13. Cell migration on ridges and cliffs

    NASA Astrophysics Data System (ADS)

    Driscoll, Meghan; McCann, Colin; Kopace, Rael; Watts, John; Homan, Tess; Losert, Wolfgang

    2009-03-01

    The amoeba Dictyostelium discoideum is a model system for the study of cellular migration, an important physiological process that occurs in embryonic development, wound healing, and cancer metastasis. We study the motion of D. discoideum on surfaces with various topographies, particularly those that affect the direction of cellular migration. Topographical features, such as ridges and cliffs, were fabricated using multiphoton absorption polymerization. As the cells encountered these topographical features, we tracked their overall motions and shapes, as well as the locations and intensities of certain intracellular signals. We found that when cells undergoing chemokinesis, random migration in response to a chemical signal, encounter a ridge, they tend to move along that ridge, even if the ridge is shorter than the cell. When cells undergoing chemotaxis, directed migration in response to a chemical signal, are directed off of a cliff, they do not fall off the cliff. Instead, they search for new attachment points, eventually change direction, and continue moving along the edge of the cliff. Both ridges and cliffs affect more than just the motion of a cell; they also affect its shape.

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

    PubMed Central

    Wen, Xi; Jin, Tian; Xu, Xuehua

    2016-01-01

    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. PMID:27684322

  15. Cell Blebbing in Confined Microfluidic Environments

    PubMed Central

    Ibo, Markela; Srivastava, Vasudha; Robinson, Douglas N.; Gagnon, Zachary R.

    2016-01-01

    Migrating cells can extend their leading edge by forming myosin-driven blebs and F-actin-driven pseudopods. When coerced to migrate in resistive environments, Dictyostelium cells switch from using predominately pseudopods to blebs. Bleb formation has been shown to be chemotactic and can be influenced by the direction of the chemotactic gradient. In this study, we determine the blebbing responses of developed cells of Dictyostelium discoideum to cAMP gradients of varying steepness produced in microfluidic channels with different confining heights, ranging between 1.7 μm and 3.8 μm. We show that microfluidic confinement height, gradient steepness, buffer osmolarity and Myosin II activity are important factors in determining whether cells migrate with blebs or with pseudopods. Dictyostelium cells were observed migrating within the confines of microfluidic gradient channels. When the cAMP gradient steepness is increased from 0.7 nM/μm to 20 nM/μm, cells switch from moving with a mixture of blebs and pseudopods to moving only using blebs when chemotaxing in channels with confinement heights less than 2.4 μm. Furthermore, the size of the blebs increases with gradient steepness and correlates with increases in myosin-II localization at the cell cortex. Reduction of intracellular pressure by high osmolarity buffer or inhibition of myosin-II by blebbistatin leads to a decrease in bleb formation and bleb size. Together, our data reveal that the protrusion type formed by migrating cells can be influenced by the channel height and the steepness of the cAMP gradient, and suggests that a combination of confinement-induced myosin-II localization and cAMP-regulated cortical contraction leads to increased intracellular fluid pressure and bleb formation. PMID:27706201

  16. Heteromeric p97/p97R155C complexes induce dominant negative changes in wild-type and autophagy 9-deficient Dictyostelium strains.

    PubMed

    Arhzaouy, Khalid; Strucksberg, Karl-Heinz; Tung, Sze Man; Tangavelou, Karthikeyan; Stumpf, Maria; Faix, Jan; Schröder, Rolf; Clemen, Christoph S; Eichinger, Ludwig

    2012-01-01

    Heterozygous mutations in the human VCP (p97) gene cause autosomal-dominant IBMPFD (inclusion body myopathy with early onset Paget's disease of bone and frontotemporal dementia), ALS14 (amyotrophic lateral sclerosis with or without frontotemporal dementia) and HSP (hereditary spastic paraplegia). Most prevalent is the R155C point mutation. We studied the function of p97 in the social amoeba Dictyostelium discoideum and have generated strains that ectopically express wild-type (p97) or mutant p97 (p97(R155C)) fused to RFP in AX2 wild-type and autophagy 9 knock-out (ATG9(KO)) cells. Native gel electrophoresis showed that both p97 and p97(R155C) assemble into hexamers. Co-immunoprecipitation studies revealed that endogenous p97 and p97(R155C)-RFP form heteromers. The mutant strains displayed changes in cell growth, phototaxis, development, proteasomal activity, ubiquitinylated proteins, and ATG8(LC3) indicating mis-regulation of multiple essential cellular processes. Additionally, immunofluorescence analysis revealed an increase of protein aggregates in ATG9(KO)/p97(R155C)-RFP and ATG9(KO) cells. They were positive for ubiquitin in both strains, however, solely immunoreactive for p97 in the ATG9(KO) mutant. A major finding is that the expression of p97(R155C)-RFP in the ATG9(KO) strain partially or fully rescued the pleiotropic phenotype. We also observed dose-dependent effects of p97 on several cellular processes. Based on findings in the single versus the double mutants we propose a novel mode of p97 interaction with the core autophagy protein ATG9 which is based on mutual inhibition.

  17. Biochemical and Structural Characterizations of Two Dictyostelium Cellobiohydrolases from the Amoebozoa Kingdom Reveal a High Level of Conservation between Distant Phylogenetic Trees of Life

    PubMed Central

    Hobdey, Sarah E.; Knott, Brandon C.; Haddad Momeni, Majid; Taylor, Larry E.; Borisova, Anna S.; Podkaminer, Kara K.; VanderWall, Todd A.; Himmel, Michael E.; Decker, Stephen R.

    2016-01-01

    ABSTRACT Glycoside hydrolase family 7 (GH7) cellobiohydrolases (CBHs) are enzymes commonly employed in plant cell wall degradation across eukaryotic kingdoms of life, as they provide significant hydrolytic potential in cellulose turnover. To date, many fungal GH7 CBHs have been examined, yet many questions regarding structure-activity relationships in these important natural and commercial enzymes remain. Here, we present the crystal structures and a biochemical analysis of two GH7 CBHs from social amoeba: Dictyostelium discoideum Cel7A (DdiCel7A) and Dictyostelium purpureum Cel7A (DpuCel7A). DdiCel7A and DpuCel7A natively consist of a catalytic domain and do not exhibit a carbohydrate-binding module (CBM). The structures of DdiCel7A and DpuCel7A, resolved to 2.1 Å and 2.7 Å, respectively, are homologous to those of other GH7 CBHs with an enclosed active-site tunnel. Two primary differences between the Dictyostelium CBHs and the archetypal model GH7 CBH, Trichoderma reesei Cel7A (TreCel7A), occur near the hydrolytic active site and the product-binding sites. To compare the activities of these enzymes with the activity of TreCel7A, the family 1 TreCel7A CBM and linker were added to the C terminus of each of the Dictyostelium enzymes, creating DdiCel7ACBM and DpuCel7ACBM, which were recombinantly expressed in T. reesei. DdiCel7ACBM and DpuCel7ACBM hydrolyzed Avicel, pretreated corn stover, and phosphoric acid-swollen cellulose as efficiently as TreCel7A when hydrolysis was compared at their temperature optima. The Ki of cellobiose was significantly higher for DdiCel7ACBM and DpuCel7ACBM than for TreCel7A: 205, 130, and 29 μM, respectively. Taken together, the present study highlights the remarkable degree of conservation of the activity of these key natural and industrial enzymes across quite distant phylogenetic trees of life. IMPORTANCE GH7 CBHs are among the most important cellulolytic enzymes both in nature and for emerging industrial applications for

  18. Quantifying and controlling collective motion in externally guided cells

    NASA Astrophysics Data System (ADS)

    Parker, Joshua; Guven, Can; Wang, Chenlu; Ott, Edward; Losert, Wolfgang

    2015-03-01

    Many motile cells use chemical signals to coordinate their motion to aid in performing a larger task, be it healing a wound or aggregating to form a spore. This coordination can vary from subtle variations in overall alignment to broad, visibly structured patterns. Of particular interest of study are two organisms We introduce a model for motion towards a chemical signal and study these spatio-temporal correlations in the context of autocrine relay, such as seen in Dictyostelium discoideum, where we demonstrate that adhesion and chemical degradation both enhance visible ``streaming'' structures. We also study a model of paracrine signal relay relevant to human neutrophil migration and demonstrate how temporally varying chemical signals can be used to coordinate cell migration. We discuss both of these results in the context of their relevance to the survival of the organism and highlight future experimental tests.

  19. Exploitation of other social amoebae by Dictyostelium caveatum.

    PubMed

    Nizak, Clément; Fitzhenry, Robert J; Kessin, Richard H

    2007-02-14

    Dictyostelium amoebae faced with starvation trigger a developmental program during which many cells aggregate and form fruiting bodies that consist of a ball of spores held aloft by a thin stalk. This developmental strategy is open to several forms of exploitation, including the remarkable case of Dictyostelium caveatum, which, even when it constitutes 1/10(3) of the cells in an aggregate, can inhibit the development of the host and eventually devour it. We show that it accomplishes this feat by inhibiting a region of cells, called the tip, which organizes the development of the aggregate into a fruiting body. We use live-cell microscopy to define the D. caveatum developmental cycle and to show that D. caveatum amoebae have the capacity to ingest amoebae of other Dictyostelid species, but do not attack each other. The block in development induced by D. caveatum does not affect the expression of specific markers of prespore cell or prestalk cell differentiation, but does stop the coordinated cell movement leading to tip formation. The inhibition mechanism involves the constitutive secretion of a small molecule by D. caveatum and is reversible. Four Dictyostelid species were inhibited in their development, while D. caveatum is not inhibited by its own compound(s). D. caveatum has evolved a predation strategy to exploit other members of its genus, including mechanisms of developmental inhibition and specific phagocytosis.

  20. Video-Rate Confocal Microscopy for Single-Molecule Imaging in Live Cells and Superresolution Fluorescence Imaging

    PubMed Central

    Lee, Jinwoo; Miyanaga, Yukihiro; Ueda, Masahiro; Hohng, Sungchul

    2012-01-01

    There is no confocal microscope optimized for single-molecule imaging in live cells and superresolution fluorescence imaging. By combining the swiftness of the line-scanning method and the high sensitivity of wide-field detection, we have developed a, to our knowledge, novel confocal fluorescence microscope with a good optical-sectioning capability (1.0 μm), fast frame rates (<33 fps), and superior fluorescence detection efficiency. Full compatibility of the microscope with conventional cell-imaging techniques allowed us to do single-molecule imaging with a great ease at arbitrary depths of live cells. With the new microscope, we monitored diffusion motion of fluorescently labeled cAMP receptors of Dictyostelium discoideum at both the basal and apical surfaces and obtained superresolution fluorescence images of microtubules of COS-7 cells at depths in the range 0–85 μm from the surface of a coverglass. PMID:23083712

  1. Proximity-Dependent Biotin Identification (BioID) in Dictyostelium Amoebae.

    PubMed

    Batsios, Petros; Meyer, Irene; Gräf, Ralph

    2016-01-01

    The identification of a bona fide lamin-like protein in Dictyostelium made this lower eukaryote an attractive model organism to study evolutionarily conserved nuclear envelope (NE) proteins important for nuclear organization and human laminopathies. Proximity-dependent biotin identification (BioID), reported by Roux and colleagues, is a powerful discovery tool for lamin-associated proteins. In this method, living cells express a bait protein (e.g., lamin) fused to an R118G-mutated version of BirA, an Escherichia coli biotinylase. In the presence of biotin, BirA-R118G biotinylates target proteins in close proximity in vivo, which are purified using streptavidin and identified by immunoblotting or mass spectrometry. We adapted the BioID method for use in Dictyostelium amoebae. The protocols described here successfully revealed Dictyostelium lamin-like protein NE81 proximity to Sun1, a conserved inner nuclear membrane protein.

  2. X-ray propagation microscopy of biological cells using waveguides as a quasipoint source

    NASA Astrophysics Data System (ADS)

    Giewekemeyer, K.; Krüger, S. P.; Kalbfleisch, S.; Bartels, M.; Beta, C.; Salditt, T.

    2011-02-01

    We have used x-ray waveguides as highly confining optical elements for nanoscale imaging of unstained biological cells using the simple geometry of in-line holography. The well-known twin-image problem is effectively circumvented by a simple and fast iterative reconstruction. The algorithm which combines elements of the classical Gerchberg-Saxton scheme and the hybrid-input-output algorithm is optimized for phase-contrast samples, well-justified for imaging of cells at multi-keV photon energies. The experimental scheme allows for a quantitative phase reconstruction from a single holographic image without detailed knowledge of the complex illumination function incident on the sample, as demonstrated for freeze-dried cells of the eukaryotic amoeba Dictyostelium discoideum. The accessible resolution range is explored by simulations, indicating that resolutions on the order of 20 nm are within reach applying illumination times on the order of minutes at present synchrotron sources.

  3. Inversely correlated cycles in speed and turning in an ameba: an oscillatory model of cell locomotion.

    PubMed Central

    Shenderov, A D; Sheetz, M P

    1997-01-01

    Previous biophysical models of ameboid crawling have described cell movement in terms of a persistent random walk. Speed and orientation were treated in the latter model as independent and temporally homogeneous stochastic processes. We show here that, at least in the case of Dictyostelium discoideum, both speed control and reorientation processes involve a deterministic, periodic component. We also show that the processes are synchronized and negatively correlated, as was suggested by earlier findings. That is, increased turning correlates with periods of slow movement. Therefore, previous models are inconsistent with the behavior of cells. Using a heuristic approach, we have developed a mathematical model that describes the statistical properties of the cell's velocity and movement of its centroid. Our observations and the model are consistent with the phenomenological description of ameboid motility as a cyclic process of pseudopod extension and retraction. Images FIGURE 2 FIGURE 3 FIGURE 5 FIGURE 6 FIGURE 7 PMID:9129842

  4. X-ray propagation microscopy of biological cells using waveguides as a quasipoint source

    SciTech Connect

    Giewekemeyer, K.; Krueger, S. P.; Kalbfleisch, S.; Bartels, M.; Salditt, T.; Beta, C.

    2011-02-15

    We have used x-ray waveguides as highly confining optical elements for nanoscale imaging of unstained biological cells using the simple geometry of in-line holography. The well-known twin-image problem is effectively circumvented by a simple and fast iterative reconstruction. The algorithm which combines elements of the classical Gerchberg-Saxton scheme and the hybrid-input-output algorithm is optimized for phase-contrast samples, well-justified for imaging of cells at multi-keV photon energies. The experimental scheme allows for a quantitative phase reconstruction from a single holographic image without detailed knowledge of the complex illumination function incident on the sample, as demonstrated for freeze-dried cells of the eukaryotic amoeba Dictyostelium discoideum. The accessible resolution range is explored by simulations, indicating that resolutions on the order of 20 nm are within reach applying illumination times on the order of minutes at present synchrotron sources.

  5. Neurofibromin controls macropinocytosis and phagocytosis in Dictyostelium

    PubMed Central

    Bloomfield, Gareth; Traynor, David; Sander, Sophia P; Veltman, Douwe M; Pachebat, Justin A; Kay, Robert R

    2015-01-01

    Cells use phagocytosis and macropinocytosis to internalise bulk material, which in phagotrophic organisms supplies the nutrients necessary for growth. Wildtype Dictyostelium amoebae feed on bacteria, but for decades laboratory work has relied on axenic mutants that can also grow on liquid media. We used forward genetics to identify the causative gene underlying this phenotype. This gene encodes the RasGAP Neurofibromin (NF1). Loss of NF1 enables axenic growth by increasing fluid uptake. Mutants form outsized macropinosomes which are promoted by greater Ras and PI3K activity at sites of endocytosis. Relatedly, NF1 mutants can ingest larger-than-normal particles using phagocytosis. An NF1 reporter is recruited to nascent macropinosomes, suggesting that NF1 limits their size by locally inhibiting Ras signalling. Our results link NF1 with macropinocytosis and phagocytosis for the first time, and we propose that NF1 evolved in early phagotrophs to spatially modulate Ras activity, thereby constraining and shaping their feeding structures. DOI: http://dx.doi.org/10.7554/eLife.04940.001 PMID:25815683

  6. PP2A/B56 and GSK3/Ras suppress PKB activity during Dictyostelium chemotaxis.

    PubMed

    Rodriguez Pino, Marbelys; Castillo, Boris; Kim, Bohye; Kim, Lou W

    2015-12-01

    We have previously shown that the Dictyostelium protein phosphatase 2A regulatory subunit B56, encoded by psrA, modulates Dictyostelium cell differentiation through negatively affecting glycogen synthase kinase 3 (GSK3) function. Our follow-up research uncovered that B56 preferentially associated with GDP forms of RasC and RasD, but not with RasG in vitro, and psrA(-) cells displayed inefficient activation of multiple Ras species, decreased random motility, and inefficient chemotaxis toward cAMP and folic acid gradient. Surprisingly, psrA(-) cells displayed aberrantly high basal and poststimulus phosphorylation of Dictyostelium protein kinase B (PKB) kinase family member PKBR1 and PKB substrates. Expression of constitutively active Ras mutants or inhibition of GSK3 in psrA(-) cells increased activities of both PKBR1 and PKBA, but only the PKBR1 activity was increased in wild-type cells under the equivalent conditions, indicating that either B56- or GSK3-mediated suppressive mechanism is sufficient to maintain low PKBA activity, but both mechanisms are necessary for suppressing PKBR1. Finally, cells lacking RasD or RasC displayed normal PKBR1 regulation under GSK3-inhibiting conditions, indicating that RasC or RasD proteins are essential for GSK3-mediated PKBR1 inhibition. In summary, B56 constitutes inhibitory circuits for PKBA and PKBR1 and thus heavily affects Dictyostelium chemotaxis.

  7. PP2A/B56 and GSK3/Ras suppress PKB activity during Dictyostelium chemotaxis

    PubMed Central

    Rodriguez Pino, Marbelys; Castillo, Boris; Kim, Bohye; Kim, Lou W.

    2015-01-01

    We have previously shown that the Dictyostelium protein phosphatase 2A regulatory subunit B56, encoded by psrA, modulates Dictyostelium cell differentiation through negatively affecting glycogen synthase kinase 3 (GSK3) function. Our follow-up research uncovered that B56 preferentially associated with GDP forms of RasC and RasD, but not with RasG in vitro, and psrA− cells displayed inefficient activation of multiple Ras species, decreased random motility, and inefficient chemotaxis toward cAMP and folic acid gradient. Surprisingly, psrA− cells displayed aberrantly high basal and poststimulus phosphorylation of Dictyostelium protein kinase B (PKB) kinase family member PKBR1 and PKB substrates. Expression of constitutively active Ras mutants or inhibition of GSK3 in psrA− cells increased activities of both PKBR1 and PKBA, but only the PKBR1 activity was increased in wild-type cells under the equivalent conditions, indicating that either B56- or GSK3-mediated suppressive mechanism is sufficient to maintain low PKBA activity, but both mechanisms are necessary for suppressing PKBR1. Finally, cells lacking RasD or RasC displayed normal PKBR1 regulation under GSK3-inhibiting conditions, indicating that RasC or RasD proteins are essential for GSK3-mediated PKBR1 inhibition. In summary, B56 constitutes inhibitory circuits for PKBA and PKBR1 and thus heavily affects Dictyostelium chemotaxis. PMID:26424797

  8. Collective Chemotactic Cell Movement; a Key Mechanism of Development and Morphogenesis

    NASA Astrophysics Data System (ADS)

    Weijer, Cornelis

    2011-03-01

    We investigate the molecular mechanisms by which cells produce and detect chemotactic signals and translate this information in directed movement up or down chemical gradients in the social amoebae Dictyostelium discoideum, and during gastrulation in the chick embryo. Investigation of Dictyostelium mutants with changes in cAMP cell-cell signalling dynamics and chemotaxis, show how cellular heterogeneity in signalling dynamics and polarised activation of the actin-myosin cytoskeleton drive aggregation, cell sorting, slug formation and migration. Chemotactic cell movement also plays a critical role during gastrulation in the chick embryo a model for amniote development. We suggest that epiblast cell movement during the formation of the primitive streak as well as the movement of the mesoderm cells after their ingression through the streak is controlled by a combination of attractive and repulsive guidance cues. We use computer models explore signalling and cell movement interact to give rise to emergent phenomena at the tissue and organism level such as pattern formation and morphogenesis.

  9. A 27,000-D core of the Dictyostelium 34,000-D protein retains Ca(2+)- regulated actin cross-linking but lacks bundling activity

    PubMed Central

    1993-01-01

    Actin cross-linking proteins are important for formation of isotropic F- actin networks and anisotropic bundles of filaments in the cytoplasm of eucaryotic cells. A 34,000-D protein from the cellular slime mold Dictyostelium discoideum mediates formation of actin bundles in vitro, and is specifically incorporated into filopodia. The actin cross- linking activity of this protein is inhibited by the presence of micromolar calcium. A 27,000-D fragment obtained by digestion with alpha-chymotrypsin lacks the amino-terminal six amino acids and the carboxyl-terminal 7,000 D of the intact polypeptide. The 27,000-D fragment retains F-actin binding activity assessed by cosedimentation assays and by 125I-[F-actin] blot overlay technique, F-actin cross- linking activity as assessed by viscometry, and calcium binding activity. Ultrastructural analyses indicate that the 27,000-D fragment is deficient in the bundling activity characteristic of the intact 34,000-D protein. Actin filaments are aggregated into microdomains but not bundle in the presence of the 27,000-D fragment. A polarized light scattering assay was used to demonstrate that the 34,000-D protein increases the orientational correlation among F-actin filaments. The 27,000-D fragment does not increase the orientation of the actin filaments as assessed by this technique. A terminal segment(s) of the 34,000-D protein, lacking in the 27,000-D fragment, contributes significantly to the ability to cross-link actin filaments into bundles. PMID:8436589

  10. Experimental Insights into Collective Effects in Eukaryotic Cell Proliferation in Dilute Suspensions

    NASA Astrophysics Data System (ADS)

    Franck, Carl; Segota, Igor; Strandburg-Peshkin, Ariana; Zhou, Xiao-Qiao S.; Rachakonda, Archana; Yavitt, Benjamin; Lussenhop, Catherine J.; Lee, Sungsu; Tharratt, Kevin; Deshmukh, Amrish; Sebesta, Elisabeth; Zhang, Myron; Lau, Sharon; Bennedsen, Sarah; Franck, David; Fernando, Viyath; Oh, Junseok

    2013-03-01

    Physicists can look to dilute suspensions of apparently solitary cells in suspension for elegant realizations of multicellular behavior. In contrast to our earlier work (Phys. Rev. E v. 77, 041905 (2008)) with the amoeba Dictyostelium discoideum we are discovering that the vital intercellular communications responsible for the well-known but poorly understood slow to fast transition in a growing culture as a function of time might be due to the passage of chemical messages between transient cell clusters or throughout the entire system as opposed to binary collisions. In considering the observed variation in proliferation rates we have been surprised to discover that for best growth cultures are much more dependent on incubator geometry than previously suspected.

  11. Fast-crawling cell types migrate to avoid the direction of periodic substratum stretching.

    PubMed

    Okimura, Chika; Ueda, Kazuki; Sakumura, Yuichi; Iwadate, Yoshiaki

    2016-07-03

    To investigate the relationship between mechanical stimuli from substrata and related cell functions, one of the most useful techniques is the application of mechanical stimuli via periodic stretching of elastic substrata. In response to this stimulus, Dictyostelium discoideum cells migrate in a direction perpendicular to the stretching direction. The origins of directional migration, higher migration velocity in the direction perpendicular to the stretching direction or the higher probability of a switch of migration direction to perpendicular to the stretching direction, however, remain unknown. In this study, we applied periodic stretching stimuli to neutrophil-like differentiated HL-60 cells, which migrate perpendicular to the direction of stretch. Detailed analysis of the trajectories of HL-60 cells and Dictyostelium cells obtained in a previous study revealed that the higher probability of a switch of migration direction to that perpendicular to the direction of stretching was the main cause of such directional migration. This directional migration appears to be a strategy adopted by fast-crawling cells in which they do not migrate faster in the direction they want to go, but migrate to avoid a direction they do not want to go.

  12. Identification of four candidate cGMP targets in Dictyostelium

    PubMed Central

    Goldberg, Jonathan M.; Bosgraaf, Leonard; Van Haastert, Peter J. M.; Smith, Janet L.

    2002-01-01

    In Dictyostelium, a transient increase in intracellular cGMP is important for cytoskeletal rearrangements during chemotaxis. There must be cGMP-binding proteins in Dictyostelium that regulate key cytoskeletal components after treatment with chemoattractants, but to date, no such proteins have been identified. Using a bioinformatics approach, we have found four candidate cGMP-binding proteins (GbpA–D). GbpA and -B have two tandem cGMP-binding sites downstream of a metallo β-lactamase domain, a superfamily that includes cAMP phosphodiesterases. GbpC contains the following nine domains (in order): leucine-rich repeats, Ras, MEK kinase, Ras guanine nucleotide exchange factor N-terminal (RasGEF-N), DEP, RasGEF, cGMP-binding, GRAM, and a second cGMP-binding domain. GbpD is related to GbpC, but is much shorter; it begins with the RasGEF-N domain, and lacks the DEP domain. Disruption of the gbpC gene results in loss of all high-affinity cGMP-binding activity present in the soluble cellular fraction. GbpC mRNA levels increase dramatically 8 h after starvation is initiated. GbpA, -B, and -D mRNA levels show less dramatic changes, with gbpA mRNA levels highest 4 h into starvation, gbpB mRNA levels highest in vegetative cells, and gbpD levels highest at 8 h. The identification of these genes is the first step in a molecular approach to studying downstream effects of cGMP signaling in Dictyostelium. PMID:12011437

  13. Dictyostelium uses ether-linked inositol phospholipids for intracellular signalling.

    PubMed

    Clark, Jonathan; Kay, Robert R; Kielkowska, Anna; Niewczas, Izabella; Fets, Louise; Oxley, David; Stephens, Len R; Hawkins, Phillip T

    2014-10-01

    Inositol phospholipids are critical regulators of membrane biology throughout eukaryotes. The general principle by which they perform these roles is conserved across species and involves binding of differentially phosphorylated inositol head groups to specific protein domains. This interaction serves to both recruit and regulate the activity of several different classes of protein which act on membrane surfaces. In mammalian cells, these phosphorylated inositol head groups are predominantly borne by a C38:4 diacylglycerol backbone. We show here that the inositol phospholipids of Dictyostelium are different, being highly enriched in an unusual C34:1e lipid backbone, 1-hexadecyl-2-(11Z-octadecenoyl)-sn-glycero-3-phospho-(1'-myo-inositol), in which the sn-1 position contains an ether-linked C16:0 chain; they are thus plasmanylinositols. These plasmanylinositols respond acutely to stimulation of cells with chemoattractants, and their levels are regulated by PIPKs, PI3Ks and PTEN. In mammals and now in Dictyostelium, the hydrocarbon chains of inositol phospholipids are a highly selected subset of those available to other phospholipids, suggesting that different molecular selectors are at play in these organisms but serve a common, evolutionarily conserved purpose.

  14. Dictyostelium Nramp1, which is structurally and functionally similar to mammalian DMT1 transporter, mediates phagosomal iron efflux.

    PubMed

    Buracco, Simona; Peracino, Barbara; Cinquetti, Raffaella; Signoretto, Elena; Vollero, Alessandra; Imperiali, Francesca; Castagna, Michela; Bossi, Elena; Bozzaro, Salvatore

    2015-09-01

    The Nramp (Slc11) protein family is widespread in bacteria and eukaryotes, and mediates transport of divalent metals across cellular membranes. The social amoeba Dictyostelium discoideum has two Nramp proteins. Nramp1, like its mammalian ortholog (SLC11A1), is recruited to phagosomal and macropinosomal membranes, and confers resistance to pathogenic bacteria. Nramp2 is located exclusively in the contractile vacuole membrane and controls, synergistically with Nramp1, iron homeostasis. It has long been debated whether mammalian Nramp1 mediates iron import or export from phagosomes. By selectively loading the iron-chelating fluorochrome calcein in macropinosomes, we show that Dictyostelium Nramp1 mediates iron efflux from macropinosomes in vivo. To gain insight in ion selectivity and the transport mechanism, the proteins were expressed in Xenopus oocytes. Using a novel assay with calcein, and electrophysiological and radiochemical assays, we show that Nramp1, similar to rat DMT1 (also known as SLC11A2), transports Fe(2+) and manganese, not Fe(3+) or copper. Metal ion transport is electrogenic and proton dependent. By contrast, Nramp2 transports only Fe(2+) in a non-electrogenic and proton-independent way. These differences reflect evolutionary divergence of the prototypical Nramp2 protein sequence compared to the archetypical Nramp1 and DMT1 proteins.

  15. Ras activation and symmetry breaking during Dictyostelium chemotaxis.

    PubMed

    Kortholt, Arjan; Keizer-Gunnink, Ineke; Kataria, Rama; Van Haastert, Peter J M

    2013-10-01

    Central to chemotaxis is the molecular mechanism by which a shallow spatial gradient of chemoattractant induces symmetry breaking of activated signaling molecules. Previously, we have used Dictyostelium mutants to investigate the minimal requirements for chemotaxis, and identified a basal signaling module providing activation of Ras and F-actin at the leading edge. Here, we show that Ras activation after application of a pipette releasing the chemoattractant cAMP has three phases, each depending on specific guanine-nucleotide-exchange factors (GEFs). Initially a transient activation of Ras occurs at the entire cell boundary, which is proportional to the local cAMP concentrations and therefore slightly stronger at the front than in the rear of the cell. This transient Ras activation is present in gα2 (gpbB)-null cells but not in gβ (gpbA)-null cells, suggesting that Gβγ mediates the initial activation of Ras. The second phase is symmetry breaking: Ras is activated only at the side of the cell closest to the pipette. Symmetry breaking absolutely requires Gα2 and Gβγ, but not the cytoskeleton or four cAMP-induced signaling pathways, those dependent on phosphatidylinositol (3,4,5)-triphosphate [PtdIns(3,4,5)P3], cGMP, TorC2 and PLA2. As cells move in the gradient, the crescent of activated Ras in the front half of the cell becomes confined to a small area at the utmost front of the cell. Confinement of Ras activation leads to cell polarization, and depends on cGMP formation, myosin and F-actin. The experiments show that activation, symmetry breaking and confinement of Ras during Dictyostelium chemotaxis uses different G-protein subunits and a multitude of Ras GEFs and GTPase-activating proteins (GAPs).

  16. Adenylyl cyclase G, an osmosensor controlling germination of Dictyostelium spores.

    PubMed

    van Es, S; Virdy, K J; Pitt, G S; Meima, M; Sands, T W; Devreotes, P N; Cotter, D A; Schaap, P

    1996-09-27

    Dictyostelium cells express a G-protein-coupled adenylyl cyclase, ACA, during aggregation and an atypical adenylyl cyclase, ACG, in mature spores. The ACG gene was disrupted by homologous recombination. acg- cells developed into normal fruiting bodies with viable spores, but spore germination was no longer inhibited by high osmolarity, a fairly universal constraint for spore and seed germination. ACG activity, measured in aca-/ACG cells, was strongly stimulated by high osmolarity with optimal stimulation occurring at 200 milliosmolar. RdeC mutants, which display unrestrained protein kinase A (PKA) activity and a cell line, which overexpresses PKA under a prespore specific promoter, germinate very poorly, both at high and low osmolarity. These data indicate that ACG is an osmosensor controlling spore germination through activation of protein kinase A.

  17. Mycobacterium marinum Degrades Both Triacylglycerols and Phospholipids from Its Dictyostelium Host to Synthesise Its Own Triacylglycerols and Generate Lipid Inclusions

    PubMed Central

    2017-01-01

    During a tuberculosis infection and inside lipid-laden foamy macrophages, fatty acids (FAs) and sterols are the major energy and carbon source for Mycobacterium tuberculosis. Mycobacteria can be found both inside a vacuole and the cytosol, but how this impacts their access to lipids is not well appreciated. Lipid droplets (LDs) store FAs in form of triacylglycerols (TAGs) and are energy reservoirs of prokaryotes and eukaryotes. Using the Dictyostelium discoideum/Mycobacterium marinum infection model we showed that M. marinum accesses host LDs to build up its own intracytosolic lipid inclusions (ILIs). Here, we show that host LDs aggregate at regions of the bacteria that become exposed to the cytosol, and appear to coalesce on their hydrophobic surface leading to a transfer of diacylglycerol O-acyltransferase 2 (Dgat2)-GFP onto the bacteria. Dictyostelium knockout mutants for both Dgat enzymes are unable to generate LDs. Instead, the excess of exogenous FAs is esterified predominantly into phospholipids, inducing uncontrolled proliferation of the endoplasmic reticulum (ER). Strikingly, in absence of host LDs, M. marinum alternatively exploits these phospholipids, resulting in rapid reversal of ER-proliferation. In addition, the bacteria are unable to restrict their acquisition of lipids from the dgat1&2 double knockout leading to vast accumulation of ILIs. Recent data indicate that the presence of ILIs is one of the characteristics of dormant mycobacteria. During Dictyostelium infection, ILI formation in M. marinum is not accompanied by a significant change in intracellular growth and a reduction in metabolic activity, thus providing evidence that storage of neutral lipids does not necessarily induce dormancy. PMID:28103313

  18. Mycobacterium marinum Degrades Both Triacylglycerols and Phospholipids from Its Dictyostelium Host to Synthesise Its Own Triacylglycerols and Generate Lipid Inclusions.

    PubMed

    Barisch, Caroline; Soldati, Thierry

    2017-01-01

    During a tuberculosis infection and inside lipid-laden foamy macrophages, fatty acids (FAs) and sterols are the major energy and carbon source for Mycobacterium tuberculosis. Mycobacteria can be found both inside a vacuole and the cytosol, but how this impacts their access to lipids is not well appreciated. Lipid droplets (LDs) store FAs in form of triacylglycerols (TAGs) and are energy reservoirs of prokaryotes and eukaryotes. Using the Dictyostelium discoideum/Mycobacterium marinum infection model we showed that M. marinum accesses host LDs to build up its own intracytosolic lipid inclusions (ILIs). Here, we show that host LDs aggregate at regions of the bacteria that become exposed to the cytosol, and appear to coalesce on their hydrophobic surface leading to a transfer of diacylglycerol O-acyltransferase 2 (Dgat2)-GFP onto the bacteria. Dictyostelium knockout mutants for both Dgat enzymes are unable to generate LDs. Instead, the excess of exogenous FAs is esterified predominantly into phospholipids, inducing uncontrolled proliferation of the endoplasmic reticulum (ER). Strikingly, in absence of host LDs, M. marinum alternatively exploits these phospholipids, resulting in rapid reversal of ER-proliferation. In addition, the bacteria are unable to restrict their acquisition of lipids from the dgat1&2 double knockout leading to vast accumulation of ILIs. Recent data indicate that the presence of ILIs is one of the characteristics of dormant mycobacteria. During Dictyostelium infection, ILI formation in M. marinum is not accompanied by a significant change in intracellular growth and a reduction in metabolic activity, thus providing evidence that storage of neutral lipids does not necessarily induce dormancy.

  19. Characterization of a 1,4-{beta}-D-glucan synthase from Dictyostelium. Final technical report

    SciTech Connect

    Blanton, R.L.

    1996-02-01

    The study of cellulose biosynthesis has a long history of frustrations, false leads, and setbacks. The authors have been able to proceed further than others who have studied eukaryotic cellulose synthesis because of the high level of enzyme activity in crude membrane preparations from developing Dictyostelium cells. This has made possible experiments to study factors that influence the activity, to determine cellular localization, and to study the development regulation of the enzyme activity. In higher plants, the challenge is still to obtain highly active membrane preparations. However, they have not been able to move beyond the level of crude membranes. The high starting activity of Dictyostelium membranes gave hope that cellulose synthase activity could be purified, allowing the identification of the polypeptides involved in cellulose synthesis. The first step in the purification of a membrane-associated activity is the solubilization of the activity; this they have not yet been able to do. They have applied some of their methods developed in the study of the Dictyostelium glucan synthase to preparation of plant membranes to see if they can obtain any in vitro activity. For instance, the disruption medium, disruption methods, and assay conditions used in Dictyostelium were used to prepare plant membranes, but without obtaining significant levels of enzyme activity.

  20. Dictyostelium RasD is required for normal phototaxis, but not differentiation.

    PubMed

    Wilkins, A; Khosla, M; Fraser, D J; Spiegelman, G B; Fisher, P R; Weeks, G; Insall, R H

    2000-06-01

    RasD, a Dictyostelium homolog of mammalian Ras, is maximally expressed during the multicellular stage of development. Normal Dictyostelium aggregates are phototactic and thermotactic, moving towards sources of light and heat with great sensitivity. We show that disruption of the gene for rasD causes a near-total loss of phototaxis and thermotaxis in mutant aggregates, without obvious effects on undirected movement. Previous experiments had suggested important roles for RasD in development and cell-type determination. Surprisingly, rasD(-) cells show no obvious changes in these processes. These cells represent a novel class of phototaxis mutant, and indicate a role for a Ras pathway in the connections between stimuli and coordinated cell movement.

  1. The Dictyostelium class I myosin, MyoD, contains a novel light chain that lacks high-affinity calcium-binding sites.

    PubMed Central

    De La Roche, Marc A; Lee, Sheu-Fen; Côté, Graham P

    2003-01-01

    Dictyostelium discoideum MyoD, a long-tailed class I myosin, co-purified with two copies of a 16 kDa light chain. Sequence analysis of the MyoD light chain showed it to be a unique protein, termed MlcD, that shares 44% sequence identity with Dictyostelium calmodulin and 43% sequence identity with Acanthamoeba castellanii myosin IC light chain. MlcD comprises four EF-hands; however, EF-hands 2-4 contain mutations in key Ca2+-co-ordinating residues that would be predicted to impair Ca2+ binding. Electrospray ionization MS of MlcD in the presence of Ca2+ and La3+ showed the presence of one major and one minor metal-binding site. MlcD contains a single tryptophan residue (Trp39), the fluorescence intensity of which was quenched upon addition of Ca2+ or Mg2+, yielding apparent dissociation constants ( K'(d)) of 52 microM for Ca2+ and 450 microM for Mg2+. The low affinity of MlcD for Ca2+ indicates that it cannot function as a sensor of physiological Ca2+. Ca2+ did not affect the binding of MlcD to MyoD or to either of the two MyoD IQ (Ile-Gln) motifs. FLAG-MlcD expressed in Dictyostelium formed a complex with MyoD, but not with the two other long-tailed Dictyostelium myosin I isoenzymes, MyoB and MyoC. Through its specific association with the Ca2+-insensitive MlcD, MyoD may exhibit distinct regulatory properties that distinguish it from myosin I isoenzymes with calmodulin light chains. PMID:12826013

  2. Evidence for nucleolar subcompartments in Dictyostelium.

    PubMed

    Catalano, Andrew; O'Day, Danton H

    2015-01-24

    The nucleolus is a multifunctional nuclear compartment usually consisting of two to three subcompartments which represent stages of ribosomal biogenesis. It is linked to several human diseases including viral infections, cancer, and neurodegeneration. Dictyostelium is a model eukaryote for the study of fundamental biological processes as well as several human diseases however comparatively little is known about its nucleolus. Unlike most nucleoli it does not possess visible subcompartments at the ultrastructural level. Several recently identified nucleolar proteins in Dictyostelium leave the nucleolus after treatment with the rDNA transcription inhibitor actinomycin-D (AM-D). Different proteins exit in different ways, suggesting that previously unidentified nucleolar subcompartments may exist. The identification of nucleolar subcompartments would help to better understand the nucleolus in this model eukaryote. Here, we show that Dictyostelium nucleolar proteins nucleomorphin isoform NumA1 and Bud31 localize throughout the entire nucleolus while calcium-binding protein 4a localizes to only a portion, representing nucleolar subcompartment 1 (NoSC1). SWI/SNF complex member Snf12 localizes to a smaller area within NoSC1 representing a second nucleolar subcompartment, NoSC2. The nuclear/nucleolar localization signal KRKR from Snf12 localized GFP to NoSC2, and thus also appears to function as a nucleolar subcompartment localization signal. FhkA localizes to the nucleolar periphery displaying a similar pattern to that of Hsp32. Similarities between the redistribution patterns of Dictyostelium nucleolar proteins during nucleolar disruption as a result of either AM-D treatment or mitosis support these subcompartments. A model for the AM-D-induced redistribution patterns is proposed.

  3. A Novel Positive Selection for Identifying Cold-Sensitive Myosin II Mutants in Dictyostelium

    PubMed Central

    Patterson, B.; Spudich, J. A.

    1995-01-01

    We developed a positive selection for myosin heavy chain mutants in Dictyostelium. This selection is based on the fact that brief exposure to azide causes wild-type cells to release from the substrate, whereas myosin null cells remain adherent. This procedure assays myosin function on a time scale of minutes and has therefore allowed us to select rapid-onset cold-sensitive mutants after random chemical mutagenesis of Dictyostelium cells. We developed a rapid technique for determining which mutations lie in sequences of the myosin gene that encode the head (motor) domain and localized 27 of 34 mutants to this domain. We recovered the appropriate sequences from five of the mutants and demonstrated that they retain their cold-sensitive properties when expressed from extrachromosomal plasmids. PMID:7498732

  4. Random insertion of GFP into the cAMP-dependent protein kinase regulatory subunit from Dictyostelium discoideum.

    PubMed Central

    Biondi, R M; Baehler, P J; Reymond, C D; Véron, M

    1998-01-01

    The green fluorescent protein (GFP) is currently being used for diverse cellular biology approaches, mainly as a protein tag or to monitor gene expression. Recently it has been shown that GFP can also be used to monitor the activation of second messenger pathways by the use of fluorescence resonance energy transfer (FRET) between two different GFP mutants fused to a Ca2+sensor. We show here that GFP fusions can also be used to obtain information on regions essential for protein function. As FRET requires the two GFPs to be very close, N- or C-terminal fusion proteins will not generally produce FRET between two interacting proteins. In order to increase the probability of FRET, we decided to study the effect of random insertion of two GFP mutants into a protein of interest. We describe here a methodology for random insertion of GFP into the cAMP-dependent protein kinase regulatory subunit using a bacterial expression vector. The selection and analysis of 120 green fluorescent colonies revealed that the insertions were distributed throughout the R coding region. 14 R/GFP fusion proteins were partially purified and characterized for cAMP binding, fluorescence and ability to inhibit PKA catalytic activity. This study reveals that GFP insertion only moderately disturbed the overall folding of the protein or the proper folding of another domain of the protein, as tested by cAMP binding capacity. Furthermore, three R subunits out of 14, which harbour a GFP inserted in the cAMP binding site B, inhibit PKA catalytic subunit in a cAMP-dependent manner. Random insertion of GFP within the R subunit sets the path to develop two-component FRET with the C subunit. PMID:9776758

  5. Random insertion of GFP into the cAMP-dependent protein kinase regulatory subunit from Dictyostelium discoideum.

    PubMed

    Biondi, R M; Baehler, P J; Reymond, C D; Véron, M

    1998-11-01

    The green fluorescent protein (GFP) is currently being used for diverse cellular biology approaches, mainly as a protein tag or to monitor gene expression. Recently it has been shown that GFP can also be used to monitor the activation of second messenger pathways by the use of fluorescence resonance energy transfer (FRET) between two different GFP mutants fused to a Ca2+sensor. We show here that GFP fusions can also be used to obtain information on regions essential for protein function. As FRET requires the two GFPs to be very close, N- or C-terminal fusion proteins will not generally produce FRET between two interacting proteins. In order to increase the probability of FRET, we decided to study the effect of random insertion of two GFP mutants into a protein of interest. We describe here a methodology for random insertion of GFP into the cAMP-dependent protein kinase regulatory subunit using a bacterial expression vector. The selection and analysis of 120 green fluorescent colonies revealed that the insertions were distributed throughout the R coding region. 14 R/GFP fusion proteins were partially purified and characterized for cAMP binding, fluorescence and ability to inhibit PKA catalytic activity. This study reveals that GFP insertion only moderately disturbed the overall folding of the protein or the proper folding of another domain of the protein, as tested by cAMP binding capacity. Furthermore, three R subunits out of 14, which harbour a GFP inserted in the cAMP binding site B, inhibit PKA catalytic subunit in a cAMP-dependent manner. Random insertion of GFP within the R subunit sets the path to develop two-component FRET with the C subunit.

  6. Evidence for nucleolar subcompartments in Dictyostelium

    SciTech Connect

    Catalano, Andrew; O’Day, Danton H.

    2015-01-24

    Highlights: • Two nucleolar subcompartments (NoSC1, NoSC2) were found in Dictyostelium. • Specific nucleolar proteins localize to different nucleolar subcompartments. • Specific proteins exit NoSC1 and NoSC2 differently upon Actinomycin D treatment. • KRKR appears to function as an NoSC2 nucleolar subcompartment localization signal. - Abstract: The nucleolus is a multifunctional nuclear compartment usually consisting of two to three subcompartments which represent stages of ribosomal biogenesis. It is linked to several human diseases including viral infections, cancer, and neurodegeneration. Dictyostelium is a model eukaryote for the study of fundamental biological processes as well as several human diseases however comparatively little is known about its nucleolus. Unlike most nucleoli it does not possess visible subcompartments at the ultrastructural level. Several recently identified nucleolar proteins in Dictyostelium leave the nucleolus after treatment with the rDNA transcription inhibitor actinomycin-D (AM-D). Different proteins exit in different ways, suggesting that previously unidentified nucleolar subcompartments may exist. The identification of nucleolar subcompartments would help to better understand the nucleolus in this model eukaryote. Here, we show that Dictyostelium nucleolar proteins nucleomorphin isoform NumA1 and Bud31 localize throughout the entire nucleolus while calcium-binding protein 4a localizes to only a portion, representing nucleolar subcompartment 1 (NoSC1). SWI/SNF complex member Snf12 localizes to a smaller area within NoSC1 representing a second nucleolar subcompartment, NoSC2. The nuclear/nucleolar localization signal KRKR from Snf12 localized GFP to NoSC2, and thus also appears to function as a nucleolar subcompartment localization signal. FhkA localizes to the nucleolar periphery displaying a similar pattern to that of Hsp32. Similarities between the redistribution patterns of Dictyostelium nucleolar proteins during

  7. Dynamics of clathrin-mediated endocytosis and its requirement for organelle biogenesis in Dictyostelium.

    PubMed

    Macro, Laura; Jaiswal, Jyoti K; Simon, Sanford M

    2012-12-01

    The protein clathrin mediates one of the major pathways of endocytosis from the extracellular milieu and plasma membrane. In single-cell eukaryotes, such as Saccharomyces cerevisiae, the gene encoding clathrin is not an essential gene, raising the question of whether clathrin conveys specific advantages for multicellularity. Furthermore, in contrast to mammalian cells, endocytosis in S. cerevisiae is not dependent on either clathrin or adaptor protein 2 (AP2), an endocytic adaptor molecule. In this study, we investigated the requirement for components of clathrin-mediated endocytosis (CME) in another unicellular organism, the amoeba Dictyostelium. We identified a heterotetrameric AP2 complex in Dictyostelium that is similar to that which is found in higher eukaryotes. By simultaneously imaging fluorescently tagged clathrin and AP2, we found that, similar to higher eukaryotes, these proteins colocalized to membrane puncta that move into the cell together. In addition, the contractile vacuole marker protein, dajumin-green fluorescent protein (GFP), is trafficked via the cell membrane and internalized by CME in a clathrin-dependent, AP2-independent mechanism. This pathway is distinct from other endocytic mechanisms in Dictyostelium. Our finding that CME is required for the internalization of contractile vacuole proteins from the cell membrane explains the contractile vacuole biogenesis defect in Dictyostelium cells lacking clathrin. Our results also suggest that the machinery for CME and its role in organelle maintenance appeared early during eukaryotic evolution. We hypothesize that dependence of endocytosis on specific components of the CME pathway evolved later, as demonstrated by internalization independent of AP2 function.

  8. A Phg2-Adrm1 Pathway Participates in the Nutrient-controlled Developmental Response in Dictyostelium

    PubMed Central

    Cherix, Nathalie; Froquet, Romain; Charette, Steve J.; Blanc, Cédric; Letourneur, François

    2006-01-01

    Dictyostelium amoebae grow as single cells but upon starvation they initiate multicellular development. Phg2 was characterized previously as a kinase controlling cellular adhesion and the organization of the actin cytoskeleton. Here we report that Phg2 also plays a role during the transition between growth and multicellular development, as evidenced by the fact that phg2 mutant cells can initiate development even in the presence of nutrients. Even at low cell density and in rich medium, phg2 mutant cells express discoidin, one of the earliest predevelopmental markers. Complementation studies indicate that, in addition to the kinase domain, the core region of Phg2 is involved in the initiation of development. In this region, a small domain contiguous with a previously described ras-binding domain was found to interact with the Dictyostelium ortholog of the mammalian adhesion-regulating molecule (ADRM1). In addition, adrm1 knockout cells also exhibit abnormal initiation of development. These results suggest that a Phg2-Adrm1 signaling pathway is involved in the control of the transition from growth to differentiation in Dictyostelium. Phg2 thus plays a dual role in the control of cellular adhesion and initiation of development. PMID:16987957

  9. Cell-alignment patterns in the collective migration of cells with polarized adhesion

    NASA Astrophysics Data System (ADS)

    Matsushita, Katsuyoshi

    2017-03-01

    Dictyostelium discoideum (Dd) utilizes inhomogeneities in the distribution of cell-cell adhesion molecules on cell membranes for collective cell migration. A simple example of an inhomogeneity is a front-side (leading-edge) polarization in the distribution at the early streaming stage. Experiments have shown that the polarized cell-cell adhesion induces side-by-side contact between cells [Beug et al., Nature (London) 274, 445 (1978), 10.1038/274445a0]. This result is counterintuitive, as one would expect cells to align front to front in contact with each other on the basis of front-side polarization. In this work, we theoretically examine whether front-side polarization induces side-by-side contact in collective cell migration. We construct a model for expressing cells with this polarization based on the two-dimensional cellular Potts model. By a numerical simulation with this model, we find cell-cell alignment wherein cells form lateral arrays with side-by-side contacts as observed in the experiments.

  10. Fitness tradeoffs between spores and nonaggregating cells can explain the coexistence of diverse genotypes in cellular slime molds.

    PubMed

    Tarnita, Corina E; Washburne, Alex; Martinez-Garcia, Ricardo; Sgro, Allyson E; Levin, Simon A

    2015-03-03

    Cellular slime molds, including the well-studied Dictyostelium discoideum, are amoebae whose life cycle includes both a single-cellular and a multicellular stage. To achieve the multicellular stage, individual amoebae aggregate upon starvation to form a fruiting body made of dead stalk cells and reproductive spores, a process that has been described in terms of cooperation and altruism. When amoebae aggregate they do not perfectly discriminate against nonkin, leading to chimeric fruiting bodies. Within chimeras, complex interactions among genotypes have been documented, which should theoretically reduce genetic diversity. This is however inconsistent with the great diversity of genotypes found in nature. Recent work has shown that a little-studied component of D. discoideum fitness--the loner cells that do not participate in the aggregation--can be selected for depending on environmental conditions and that, together with the spores, they could represent a bet-hedging strategy. We suggest that in all cellular slime molds the existence of loners could resolve the apparent diversity paradox in two ways. First, if loners are accounted for, then apparent genotypic skew in the spores of chimeras could simply be the result of different investments into spores versus loners. Second, in an ecosystem with multiple local environments differing in their food recovery characteristics and connected globally via weak-to-moderate dispersal, coexistence of multiple genotypes can occur. Finally, we argue that the loners make it impossible to define altruistic behavior, winners or losers, without a clear description of the ecology.

  11. Cell adhesion molecules: detection with univalent second antibody

    PubMed Central

    1980-01-01

    Identification of cell surface molecules that play a role in cell-cell adhesion (here called cell adhesion molecules) has been achieved by demonstrating the inhibitory effect of univalent antibodies that bind these molecules in an in vitro assay of cell-cell adhesion. A more convenient reagent, intact (divalent) antibody, has been avoided because it might agglutinate the cells rather than blocking cell-cell adhesion. In this report, we show that intact rabbit immunoglobulin directed against certain cell surface molecules of Dictyostelium discoideum blocks cell-cell adhesion when the in vitro assay is performed in the presence of univalent goat anti-rabbit antibody. Under appropriate experimental conditions, the univalent second antibody blocks agglutination induced by the rabbit antibody without significantly interfering with its effect on cell-cell adhesion. This method promises to be useful for screening monoclonal antibodies raised against potential cell adhesion molecules because: (a) it allows for the screening of large numbers of antibody samples without preparation of univalent fragments; and (b) it requires much less antibody because of the greater affinity of divalent antibodies for antigens. PMID:6970200

  12. Cell Shape Dynamics: From Waves to Migration

    NASA Astrophysics Data System (ADS)

    Driscoll, Meghan; McCann, Colin; Sun, Xiaoyu; Fourkas, John; Parent, Carole; Losert, Wolfgang

    2012-02-01

    We observe and quantify wave-like characteristics of amoeboid migration. Using the amoeba Dictyostelium discoideum, a model system for the study of chemotaxis, we demonstrate that cell shape changes in a wave-like manner. Cells have regions of high boundary curvature that propagate from the leading edge toward the back, usually along alternating sides of the cell. Curvature waves are easily seen in cells that do not adhere to a surface, such as cells that are electrostatically repelled from surfaces or cells that extend over the edge of micro-fabricated cliffs. Without surface contact, curvature waves travel from the leading edge to the back of a cell at ˜35 μm/min. Non-adherent myosin II null cells do not exhibit these curvature waves. At the leading edge of adherent cells, curvature waves are associated with protrusive activity. Like regions of high curvature, protrusive activity travels along the boundary in a wave-like manner. Upon contact with a surface, the waves stop moving relative to the surface, and the boundary shape thus reflects the history of protrusive motion. The wave-like character of protrusions provides a plausible mechanism for the ability of cells to both swim in viscous fluids and to navigate complex 3-D topography.