Cellular Slime Mold Dictyostelium Discoideum Research Articles

Cloning and characterization of the Dictyostelium discoideum cycloartenol synthase cDNA.

Cycloartenol synthase converts oxidosqualene to cycloartenol, the first carbocyclic intermediate en route to sterols in plants and many protists. Presented here is the first cycloartenol synthase gene identified from a protist, the cellular slime mold Dictyostelium discoideum. The cDNA encodes an 81-kDa predicted protein 50-52% identical to known higher plant cycloartenol synthases and 40-49% identical to known lanosterol synthases from fungi and mammals. The encoded protein expressed in transgenic Saccharomyces cerevisiae converted synthetic oxidosqualene to cycloartenol in vitro. This product was characterized by 1H and 13C nuclear magnetic resonance and gas chromatography-mass spectrometry. The predicted protein sequence diverges sufficiently from the known cycloartenol synthase sequences to dramatically reduce the number of residues that are candidates for the catalytic difference between cycloartenol and lanosterol formation.

Myosin II-Independent Cytokinesis in Dictyostelium. Its Mechanism and Implications.

Similar to higher animal cells, ameba cells of the cellular slime mold Dictyostelium discoideum form contractile rings containing filaments of myosin II during mitosis, and it is generally believed that contraction of these rings bisects the cells both on substrates and in suspension. In suspension, mutant cells lacking the single myosin II heavy chain gene cannot carry out cytokinesis, become large and multinucleate, and eventually lyze, supporting the idea that myosin II plays critical roles in cytokinesis. These mutant cells are however viable on substrates. Detailed analyses of these mutant cells on substrates revealed that, in addition to "classic" cytokinesis which depends on myosin II ("cytokinesis A"), Dictyostelium has two distinct, novel methods of cytokinesis, 1) attachment-assisted mitotic cleavage employed by myosin II null cells on substrates ("cytokinesis B"), and 2) cytofission, a cell cycle-independent division of adherent cells ("cytokinesis C"). Cytokinesis A, B, and C lose their function and demand fewer protein factors in this order. Cytokinesis B is of particular importance for future studies. Similar to cytokinesis A, cytokinesis B involves formation of a cleavage furrow in the equatorial region, and it may be a primitive but basic mechanism of efficiently bisecting a cell in a cell cycle-coupled manner. Analysis of large, multinucleate myosin II null cells suggested that interactions between astral microtubules and cortices positively induce polar protrusive activities in telophase. A model is proposed to explain how such polar activities drive cytokinesis B, and how cytokinesis B is coordinated with cytokinesis A in wild type cells.

Numerical aspects of discrete and continuum hybrid models in cell biology

In this paper we introduce a method of modeling which mixes continuum and discrete variables, and explain two models in cell biology that use this method. The first application deals with wound healing, more specifically the collagen alignment in scar tissue formation and the second models early aggregation in the cellular slime mold Dictyostelium discoideum. We solve these models using numerical techniques similar to the particle-in-cell method which requires that the discrete and continuum variables are interpolated one to the other. The implementational and numerical details are discussed in an informal and practical manner with particular attention given to the problem of interpolation.

JAKs and STATs in invertebrate model organisms.

Invertebrate organisms provide systems to elucidate the developmental roles of Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signaling pathways, thereby complementing research conducted with mammalian cells and animals. Components of the JAK/STAT protein pathway have been identified and characterized in the fruit fly Drosophila melanogaster and the cellular slime mold Dictyostelium discoideum. This review summarizes the molecular and genetic data obtained from these model organisms. In particular, a Drosophila JAK/STAT pathway regulates normal segmentation, cell proliferation, and differentiation, and hyperactivation of the pathway leads to tumor formation and leukemia-like defects. A Dictyostelium STAT regulates the development of stalk cells during the multicellular part of the life cycle. Future research utilizing these organisms should continue to provide insights into the roles and regulation of these proteins and their signaling pathways.

Phototaxis during the slug stage of Dictyostelium discoideum: a model study

During the slug stage, the cellular slime mould Dictyostelium discoideum moves towards light sources. We have modelled this phototactic behaviour using a hybrid cellular automata/partial differential equation model. In our model, individual amoebae are not able to measure the direction from which the light comes, and differences in light intensity do not lead to differentiation in motion velocity among the amoebae. Nevertheless, the whole slug orientates itself towards the light. This behaviour is mediated by a modification of the cyclic AMP (cAMP) waves. As an explanation for phototaxis, we propose the following mechanism, which is basically characterized by four processes: (i) light is focused on the distal side of the slug as a result of the so-called 'lens-effect'; (ii) differences in luminous intensity cause differences in NH3 concentration; (iii) NH3 alters the excitablity of the cell, and thereby the shape of the cAMP wave; and (iv) chemotaxis towards cAMP causes the slug to turn. We show that this mechanism can account for a number of other behaviours that have been observed in experiments, such as bidirectional phototaxis and the cancellation of bidirectionality by a decrease in the light intensity or the addition of charcoal to the medium.

Theoretical model for morphogenesis and cell sorting in Dictyostelium discoideum

The morphogenetic movement and cell sorting in cell aggregates from the mound stage to the migrating slug stage of the cellular slime mold Dictyostelium discoideum were studied using a mathematical model. The model postulates that the motive force generated by the cells is in equilibrium with the internal pressure and mechanical resistance. The moving boundary problem derived from the force balance equation and the continuity equation has stationary solutions in which the aggregate takes the shape of a spheroid (or an ellipse in two-dimensional space) with the pacemaker at one of its foci, moving at a constant speed. Numerical calculations in two-dimensional space showed that an irregularly shaped aggregate changes its shape to become an ellipse as it moves. Cell aggregates consisting of two cell types differing in motive force exhibit cell sorting and become elongated, suggesting the importance of prestalk/prespore differentiation in the morphogenesis of Dictyostelium.

Green fluorescent proteins with short half-lives as reporters in Dictyostelium discoideum.

We describe two modifications of the popular reporter green fluorescent protein (GFP) which have short half-lives in our system, the cellular slime mould Dictyostelium discoideum. One of these bears an N-terminal ubiquitin; this GFP was originally planned to be a substrate of the "N-end-rule" pathway, but deubiquitination does not seem to occur, and a degradation by the UFD (ubiquitin-fusion-degradation pathway seems more probable. The protein half-life is about 3-5 h. The second construct has an N-terminus derived from the L11 ribosomal protein; it is transported to the nucleus and broken down much more rapidly than the ubiquitin fusion (protein half-life about 30 min). We show examples of the use of these reporters in the study of gene expression in Dictyostelium.

Collective Motion of the Cellular Slime Molds. A Soft Interface Emerging from Motile Coupled Oscillators.

The behavior of a simple coupled oscillator model for a reaction-diffusion-chemotaxis system is examined wish respect to the morphogenesis of the cellular slime mold Dictyostelium discoideum. We explain how the intrinsic difference in the oscillation frequency of secreted chemoattractant gives rise to an anterior-posterior polarity which could drive the migration of a pseudoplasmodium. The phase locking and migration of oscillators and their implication to a phenomenological soft-interface are discussed.

A new member of the GP138 multigene family implicated in cell interactions in Dictyostelium discoideum.

The cellular slime mold Dictyostelium discoideum reproduces sexually under submerged and dark conditions. Its mating system is polymorphic and particularly interesting with respect to mechanisms of cell recognition. The cell-surface glycoprotein gp138 has been implicated in sexual cell interactions, as it was identified as a target molecule for the antibodies that block sexual cell fusion in D. discoideum. Two mutually homologous genes, GP138A and GP138B, have been cloned, but gene disruption experiments to clarify their functional relationships suggested that there is at least one more gene for gp138. Further protein analysis including peptide mapping also revealed that gp138 exists as three isoforms, DdFRP1, DdFRP2, and DdFRP3. GP138A encodes DdFRP2 and GP138B, DdFRP3, and the presence of a third gp138 gene encoding DdFRP1 was suggested. Here, we isolated and characterized a third GP138 gene, GP138C. Although the deduced amino acid sequences of GP138C matched completely with those of peptide fragments of DdFRP1 in the N-terminal half, the rest did not give complete matches. Overexpression of GP138C caused an increase in the intensity of DdFRP1, but disruption of this gene did not diminish DdFRP1. Our results indicate that GP138C encodes a protein very similar to but distinct from DdFRP1. The GP138 multigene family is thus composed of more members than previously expected, and their functional relationships are of special interest.

Analysis of the Disruption Mutant of the Oscillin Homolog Gene of Dictyostelium discoideum.

A homolog of oscillin, the Ca2+ oscillation-inducing factor of the hamster, was identified from the cellular slime mold Dictyostelium discoideum and designated Dd-oscillin. In the developmental stages of D. discoideum, the gene is expressed at the prestalk region which contains a higher concentration of cytosolic Ca2+ than the prespore region. The Dd-oscillin null strain aggregated but did not develop further when the cells were plated on non-nutrient agar at a density of 1.5x10(6) cells/cm2, showing that the Dd-oscillin gene is important for development. Since the null cells carrying the hamster oscillin gene formed fruiting body, the hamster oscillin was the homolog of Dd-oscillin as far as function is concerned. In addition, the null cells formed fruiting body in the presence of 2,5-di(tert-butyl)-1,4-hydroquinone (BHQ: a specific inhibitor of Ca2+-ATPase activity in the endoplasmic reticulum). These results suggest that Dd-oscillin will increase cytosolic Ca2+ in the cells and promote further development.

Effects of DIF-1, an Anti-Tumor Agent Isolated from Dictyostelium discoideum, on Rat Gastric Mucosal RGM-1 and Leptomeningeal Cells

DIF-1 is a putative morphogen that induces stalk cell formation in the cellular slime mold Dictyostelium discoideum. We have previously discovered that DIF-1 suppresses cell growth and induces cell differentiation in vitro in some tumor cells, and also that relatively low concentrations of DIF-1 promote retinoic acid-induced cell differentiation in the human myeloid leukemia HL-60 cells. In this study, to verify cell biological and therapeutic potential of DIF-1, we have examined whether and how DIF-1 affects normal mammalian cells in vitro, using rat leptomeningeal (RLM) cells and rat gastric mucosal RGM-1 cells. In growing phase of both cells, DIF-1 at 5–40 μM suppressed cell growth in a dose-dependent manner. High concentrations (15–40 μM) of DIF-1 were toxic to the growing cells so that the cells showed unusual morphology, but many of them were still alive even at Day 3–4. Withdrawal of DIF-1 allowed the cells to grow. In confluent phase of the cells, DIF-1 at more than 15 μM promoted medium acidification that resulted in cell death, but DIF-1 itself did not markedly affect cell viability for 3 days. DIF-1 increased [Ca2 ]i in RLM cells but did not affect β-trace secretion (an index of cell function in RLM cells). A low concentration (5 μM) of DIF-1 in combination with retinoic acid (0.1 μM) showed no marked effects on cell growth, cell viability, cell morphology and β-trace secretion in RLM cells. The present results indicate that DIF-1 may be utilized as a tool for cell biology. Also, since these concentrations of DIF-1 kill some tumor cells within 2–3 days, the present results suggest that DIF-1 might be utilized in the treatment of some sorts of cancer with a bearable degree of side-effects.

A developmentally regulated kinesin-related motor protein from Dictyostelium discoideum.

The cellular slime mold Dictyostelium discoideum is an attractive system for studying the roles of microtubule-based motility in cell development and differentiation. In this work, we report the first molecular characterization of kinesin-related proteins (KRPs) in Dictyostelium. A PCR-based strategy was used to isolate DNA fragments encoding six KRPs, several of which are induced during the developmental program that is initiated by starvation. The complete sequence of one such developmentally regulated KRP (designated K7) was determined and found to be a novel member of the kinesin superfamily. The motor domain of K7 is most similar to that of conventional kinesin, but unlike conventional kinesin, K7 is not predicted to have an extensive alpha-helical coiled-coil domain. The nonmotor domain is unusual and is rich in Asn, Gln, and Thr residues; similar sequences are found in other developmentally regulated genes in Dictyostelium. K7, expressed in Escherichia coli, supports plus end-directed microtubule motility in vitro at a speed of 0.14 micron/s, indicating that it is a bona fide motor protein. The K7 motor is found only in developing cells and reaches a peak level of expression between 12 and 16 h after starvation. By immunofluorescence microscopy, K7 localizes to a membranous perinuclear structure. To examine K7 function, we prepared a null cell line but found that these cells show no gross developmental abnormalities. However, when cultivated in the presence of wild-type cells, the K7-null cells are mostly absent from the prestalk zone of the slug. This result suggests that in a population composed largely of wild-type cells, the absence of the K7 motor protein interferes either with the ability of the cells to localize to the prestalk zone or to differentiate into prestalk cells.

Evaluation of the Teratogenic Potential of Valproic Acid Analogues in Transgenic Dictyostelium discoideum Strains

Very early during the development of new pharmaceuticals toxicological tests are most important. In addition to acute and chronic toxicity tests, it is crucial to estimate the teratogenic potential of promising drugs. We established a simple biological test system based on the cellular slime mold Dictyostelium discoideum. Under certain environmental conditions single cells of D. discoideum aggregate and undergo a relatively simple cell differentiation program, leading to the formation of stalk and spore cells. Transgenic D. discoideum strains carrying the bacterial β-galactosidase gene under the control of various developmentally regulated D. discoideum promoters were shown to be useful tools to test the teratogenic potential of valproic acid (VPA). This study describes the effects of the VPA analogues S-4-yn-VPA, R-4-yn-VPA, and 2-ethyl-4-pentynoic acid on the D. discoideum developmental system. The presence of S-4-yn-VPA during D. discoideum development resulted in a strong inhibition of spore cell differentiation, whereas stalk cell formation was less affected. The enantiomer R-4-yn-VPA as well as 2-ethyl-4-pentynoic acid had only moderate effects on D. discoideum development. The above results are consistent with data obtained in mammalian teratogenicity assays, and suggest that D. discoideum development should be investigated with a number of additional substances to provide a simple alternative for high throughput screenings of new drugs.

Steady-state force–velocity relation of ATP-dependent sliding between slime mold myosin, arranged on paramyosin filaments, and algal cell actin cables

To investigate characteristics of ATP-dependent sliding of a non-muscle cell myosin, obtained from a cellular slime mold Dictyostelium discoideum, on actin filament, we prepared hybrid thick filaments, in which Dictyostelium myosin was regularly arranged around paramyosin filaments obtained from a molluscan smooth muscle. A single to a few hybrid filaments were attached to a polystyrene bead (diameter, 4.5 μm; specific gravity, 1.5), and the filaments were made to slide on actin filament arrays (actin cables) in the internodal cell of an alga Chara corallina, mounted on the rotor of a centrifuge microscope. The filament-attached bead was observed to move with a constant velocity under a constant external load for many seconds. The steady-state force–velocity relation of Dictyostelium myosin sliding on actin cables was hyperbolic in shape except for large loads ≤0.7–0.8 P 0, being qualitatively similar to that of skeletal muscle fibres, despite a considerable variation in the number of myosin molecules interacting with actin cables. Comparison of the P–V curves between Dictyostelium myosin and muscle myosins sliding on actin cables suggests that the time of attachment to actin in a single attachment–detachment cycle is much longer in Dictyostelium myosin than in muscle myosins.

Rapid changes of nucleotide excision repair gene expression following UV-irradiation and cisplatin treatment of Dictyostelium discoideum.

Organisms use different mechanisms to detect and repair different types of DNA damage, and different species vary in their sensitivity to DNA damaging agents. The cellular slime mold Dictyostelium discoideum has long been recognized for its unusual resistance to UV and ionizing radiation. We have recently cloned three nucleotide excision repair (NER) genes from Dictyostelium , the rep B, D and E genes (the homologs of the human xeroderma pigmentosum group B, D and E genes, respectively). Each of these genes has a unique pattern of expression during the multicellular development of this organism. We have now examined the response of these genes to DNA damage. The rep B and D DNA helicase genes are rapidly and transiently induced in a dose dependent manner following exposure to both UV-light and the widely used chemotherapeutic agent cisplatin. Interestingly, the rep E mRNA level is repressed by UV but not by cisplatin, implying unique signal transduction pathways for recognizing and repairing different types of damage. Cells from all stages of growth and development display the same pattern of NER gene expression following exposure to UV-light. These results suggest that the response to UV is independent of DNA replication, and that all the factors necessary for rapid transcription of these NER genes are either stable throughout development, or are continuously synthesized. It is significant that the up-regulation of the rep B and D genes in response to UV and chemical damage has not been observed to occur in cells from other species. We suggest that this rapid expression of NER genes is at least in part responsible for the unusual resistance of Dictyostelium to DNA damage.

Differential Effects of Aminosubstituted Analogs of Hydroxy Bisphosphonates on the Growth of Dictyostelium discoideum

Replacing the hydroxyl group in the bone-binding site of three clinically useful bisphosphonates (etidronate, pamidronate, and olpadronate) by an amino group resulted in great differences in their antiresorptive potencies in vitro. In the present study, this is also shown in vivo in mice treated with the six bisphosphonates at doses of up to 16 microM/kg/day for 12 days. Because binding to bone mineral is nearly the same for all tested bisphosphonates, these findings suggest that the aminosubstitution affects the cellular action of the bisphosphonates. This was tested in the cellular slime mould Dictyostelium discoideum in which cellular effects of bisphosphonates can be examined independently of binding to bone mineral. Etidronate and its aminosubstituted analog were equipotent in inhibiting amebal growth, while pamidronate was somewhat more potent than its analog. Whereas olpadronate was a potent inhibitor of axenic growth of Dictyostelium amebae, the aminosubstitution reduced its potency drastically (IC50 12 microM and 700 microM, respectively). The similarities between the inhibitory effects of the bisphosphonates tested on bone resorption in vitro and in vivo and on the growth of Dictyostelium amebae confirm that the differences in antiresorptive potencies found reflect differences in cellular effects and suggest that bisphosphonates may bind to more than one intracellular target.

High levels of actin tyrosine phosphorylation: Correlation with the dormant state of Dictyostelium spores

ABSTRACT Upon removal of nutrients, the amoebae of the cellular slime mold Dictyostelium discoideum differentiate into dormant spores which survive starvation stress. In this study, we demonstrate that half of the actin molecules in the spores are tyrosine-phosphorylated. The phosphorylated actin is distributed around immobile crenate mitochondria and vesicles, as well as in the cytoplasm of the spores. The actin isolated from spore lysates contains phosphorylated and unphosphorylated forms at the same molar ratio as that of the original whole spore lysate. Under actin polymerizing conditions they form actin filaments and then they are completely depolymerized under actin depolymerizing conditions, indicating that tyrosine phosphorylation of actin may not prohibit actin polymerization nor stimulate depolymerization. The phosphorylation levels increase at the end of the culmination stage when spores have matured morphologically and physiologically, and reach maximum levels after an additional 12 hours of development. The levels are stable for 20 days following spore maturation, and decline to undetectable levels within the next 10 days. Spores having high levels of phosphorylation show high viability, and vice versa. Following activation of spores with nutrient medium containing spore germination promoters, the phosphorylation levels quickly decrease with a half-life of about 5 minutes. After 20 minutes spores begin to swell. At this later time, most of the phosphorylated actin already has been dephosphorylated. Also, in heat-activated spores actin dephosphorylation occurs prior to spore swelling. However, addition of phosphatase inhibitors following heat-activation, prevented spore swelling and dephosphorylation of actin. Our data indicate that the high levels of actin tyrosine phosphorylation, specific to the spore stage, may be required for maintaining dormancy to withstand starvation stress. The rapid dephosphorylation of actin leads to a reactivated dynamic actin system which participates in spore swelling, vesicle movement, and mitochondrial shape changes during the spore germination process.

High levels of actin tyrosine phosphorylation: correlation with the dormant state of Dictyostelium spores.

Upon removal of nutrients, the amoebae of the cellular slime mold Dictyostelium discoideum differentiate into dormant spores which survive starvation stress. In this study, we demonstrate that half of the actin molecules in the spores are tyrosine-phosphorylated. The phosphorylated actin is distributed around immobile crenate mitochondria and vesicles, as well as in the cytoplasm of the spores. The actin isolated from spore lysates contains phosphorylated and unphosphorylated forms at the same molar ratio as that of the original whole spore lysate. Under actin polymerizing conditions they form actin filaments and then they are completely depolymerized under actin depolymerizing conditions, indicating that tyrosine phosphorylation of actin may not prohibit actin polymerization nor stimulate depolymerization. The phosphorylation levels increase at the end of the culmination stage when spores have matured morphologically and physiologically, and reach maximum levels after an additional 12 hours of development. The levels are stable for 20 days following spore maturation, and decline to undetectable levels within the next 10 days. Spores having high levels of phosphorylation show high viability, and vice versa. Following activation of spores with nutrient medium containing spore germination promoters, the phosphorylation levels quickly decrease with a half-life of about 5 minutes. After 20 minutes spores begin to swell. At this later time, most of the phosphorylated actin already has been dephosphorylated. Also, in heat-activated spores actin dephosphorylation occurs prior to spore swelling. However, addition of phosphatase inhibitors following heat-activation, prevented spore swelling and dephosphorylation of actin. Our data indicate that the high levels of actin tyrosine phosphorylation, specific to the spore stage, may be required for maintaining dormancy to withstand starvation stress. The rapid dephosphorylation of actin leads to a reactivated dynamic actin system which participates in spore swelling, vesicle movement, and mitochondrial shape changes during the spore germination process.

Cell shape, motility and distribution of F-actin in amoebae of the mycetozoans Protostelium mycophaga and Acrasis rosea. A comparison with Dictyostelium discoideum

Summary We investigated, by a computer-assisted video analysis, by F-actin cytochemistry, and by scanning electron microscopy (SEM), the dynamic morphology, the locomotory behavior, and the actin cytoskeleton of trophic amoebae of the mycetozoans Protostelium mycophaga and Acrasis rosea, and compared them with amoebae of the cellular slime mold Dictyostelium discoideum. The three species represent protist taxa marked by different reproductive systems and by differences of amoeboid motility in the non-reproductive phase of the life cycle. Live observations and the numerical analysis of video recordings revealed characteristic traits of cell shape changes and motility in each species. The leading edge of Protostelium amoebae is dominated by a large lamellipodium that is studded with pseudodigits. We computed a mean cell size (outline area) of 498 μm2 and a mean velocity of 26.5 μm/min (centroid displacement) for this species (N = 30, as for the other organisms). Most amoebae followed a sinuous path during the observation period, which resulted in a mean standard deviation of vector direction (SDV) of 0.64, but some hardly moved at all. Amoebae of Acrasis are essentially monopodial, with lobose, smooth pseudopodia. Their mean cell size was 759 μm2 and their mean velocity 71.6 um/min. They are thus among the fastest “crawling” eucaryotic cells, which may be related to the fact that they contain no cytoplasmic microtubules. Most amoebae followed a straight path, but some made one or two abrupt turns during the observation period (SDV = 0.58). The corresponding values for Dictyostelium amoebae with filose or lobose pseudopodia were 220 μm2, 10.3 μm/min, and SDV = 0.80. In amoebae of all the three species F-actin was localized in the cell cortex, especially in hyaline pseudopodia. As seen by SEM, Protostelium amoebae had the largest area of contact with the substratum, whereas Acrasis amoebae adhered with a foot-like structure, the leading edge and the uropod being detached from the support.

A protein kinase C-like activity involved in the chemotactic response of Dictyostelium discoideum

During the developmental life cycle of the cellular slime mould Dictyostelium discoideum cells aggregate in response to pulses of extracellular cAMP. This chemotactic agent stimulates a number of signalling pathways in the cell including the activation of a phospholipase C activity leading to the transient generation of inositol 3,4,5-trisphosphate and diacylglycerol. The role of diacylglycerol in chemotactic response and development of Dictyostelium is not known. We have evidence to suggest that two protein kinase C-like enzymes exist in Dictyostelium due to the different cellular responses to two inhibitors specific for protein kinase C. One enzyme is preferentially sensitive to d- erythro-sphingosine, a diacylglycerol analogue, and is required for growth. A second is preferentially inhibited by bisindolylmaleimide GF109203X and is required for chemotaxis. We have identified protein kinase C-like kinase activity in Dictyostelium cell extracts which appears as the cells aggregate. This activity is stimulated by diacylglycerol, especially biologically relevant diacylglycerol species, and phosphorylates a peptide substrate which is an efficient substrate for mammalian protein kinase Cs. This activity is a candidate for the effector of diacylglycerol generated during the aggregative phase of Dictyostelium development and defines a role for diacylglycerol in the chemotactic response.