Motility mutants of Dictyostelium discoideum

We describe six motility mutants of Dictyostelium discoideum in this report. They were identified among a group of temperature-sensitive growth (Tsg) mutants that had been previously isolated using an enrichment for phagocytosis-defective cells. The Tsg mutants were screened for their ability to produce tracks on gold-coated cover slips, and several strains were found that were temperature-sensitive for migration in this assay. Analysis of spontaneous Tsg+ revertants of 10 migration-defective strains identified six strains that co-reverted the Tsg and track formation phenotypes. Characterization of these six strains indicated that they were defective at restrictive temperature in track formation, phagocytosis of bacteria, and pseudopodial and filopodial activity, while retaining normal rates of oxygen consumption and viability. Because they had lost this group of motile capabilities, these strains were designated motility mutants. The Tsg+ revertants of these mutants, which coordinately recovered all of the motile activities, were found at frequencies consistent with single genetic events. Analysis of the motility mutants and their revertants suggests a relationship between the motility mutations in some of these strains and genes affecting axenic growth.     

An Optical Model for Phototactic Orientation in Dictyostelium discoideum Slugs

The optical properties of an enlarged optical model of the tipof a slug (a glass tube filled with sucrose solution) of Dictyosteliumdiscoideum were studied to validate the hypothesis that theorganism orients itself with respect to lateral light usinga lens effect: Light is focussed by refraction onto the distalside of the slug where the higher "signal" compared to the frontside causes a turn toward the light. Light was effectively focusedeven when the model deviated only as little as 10° fromthe light direction. Slugs of D. discoideum strains HO596 and HO813 show bidirectionalphototaxis: They orient themselves at some angle on either sideof the incident light beam. This angle is {small tilde}80°in HO596 and {small tilde}70° in HO813. We suggest thatthis phenomenon can be explained by the optical properties ofthe slug such as absorption and scattering, shape of the tipor location and extension of the light-sensitive zone. The angleof orientation with respect to light is regulated by a balancebetween a turn toward and a turn away from the light direction. (Received October 15, 1985; Accepted February 10, 1986)     

Phototaxis genes on linkage group V in Dictyostelium discoideum

Abstract During the course of mapping and complementation analysis of phototaxis ( pho ) mutations in Dictyostelium discoideum we have assigned to linkage group V three mutant pho alleles belonging to complementation groups phoG and phoK . These are the first genetic markers with an easily recognizable phenotype to be found on this linkage group.     

Inhibition of Dictyostelium discoideum beta-glucosidase by purines.

beta-Glucosidase of Dictyostelium discoideum is inhibited by purines in the following order: adenine greater than adenosine greater than 6-methylaminopurine greater than hypoxanthine greater than inosine greater than purine greater than guanosine. Adenine inhibits activity by 50% at 1 to 2 mM. The kinetics are complex because the enzyme is stimulated by substrate and inhibited by glucose.     

Inhibition of Differentiation in Dictyostelium discoideum by Anti-Inflammatory Drugs

Evidence is presented that a panel of non-steroidal anti-inflammatory drugs inhibit both developmental gene expression and terminal cell differentiation in the slime mold Dictyostelium discoideum. Incubation of developing cells with a number of these drugs also prevents the accumulation in the cells of two lipid species which have chromatographic properties similar to authentic eicosanoids. The results raise the possibility that Dicytostelium cells synthesize eicosanoid-like lipids which are required for normal development.     

Inhibition of development in Dictyostelium discoideum by sugars.

Sugars such as glucose, maltose, and trehalose, which are metabolized by Dictyostelium discoideum and which enhance vegetative growth, inhibit the development of the slime mold at concentrations which stimulate growth maximally. They block the acquisition of aggregation competence as well as aggregation. The same sugars also inhibit the degradation of preformed glycogen ribonucleic acid, and protein, which is characteristic of development and which occurs when the amoebas are starved by incubation in dilute phosphate buffer.     

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.     

Inhibition of cell differentiation and cell cohesion by tunicamycin in Dictyostelium discoideum

The effects of tunicamycin on protein glycosylation and cell differentiation were examined during early development of Dictyostelium discoideum. Tunicamycin inhibited cell growth reversibly in liquid medium. At a concentration of 3 μg/ml, tunicamycin completely inhibited morphogenesis and cell differentiation in developing cells. These cells remained as a smooth lawn and failed to undergo chemotactic migration. The expression of EDTA-resistant contact sites was also inhibited. The inhibition by tunicamycin was reversible if cells were washed free of the drug within the first 10 hr of incubation. After 12 hr of development, cells were protected from the drug by the sheath. When cells were treated with tunicamycin during the first 10 hr of development, incorporation of [³H]mannose and [³H] fucose was inhibited by approximately 75% within 45 min while no significant inhibition of [³H]leucine incorporation was observed during the initial 3 hr of drug treatment. The inhibition of protein glycosylation was further evidenced by the reduction in number of glycoproteins “stained” with ¹²⁵I-labelled con A. A number of developmentally regulated high-molecular-weight glycoproteins, including the contact site A glycoprotein (gp80), were undetectable when cells were labelled with [³H]fucose in the presence of tunicamycin. It is therefore evident that glycoproteins with N-glycosidically linked carbohydrate moieties may play a crucial role in intercellular cohesiveness and early development of D. discoideum.     

Ultraviolet Irradiation of the Vegetative Cells of Dictyostelium discoideum

Experiments on the effect of ultraviolet (UV) light on the survival of vegetative Dictyostelium discoideum cells indicate that this is a relatively UV-resistant organism. Several factors suggest the presence of some type of repair process. Experiments to test for liquid-holding recovery and simple photoreactivation yielded negative results. Acriflavine and caffeine were utilized to possibly interfere with dark repair. Acriflavine produced no UV sensitization, but caffeine did cause a concentration-dependent decrease in survival of irradiated cells. When UV-irradiated cells were illuminated with photoreactivating light while suspended in caffeine, the survival increased above that for cells treated with caffeine alone, suggesting an overlap between lesions repaired by photorepair and dark repair. Growth experiments showed that UV light induced a dose-dependent division delay, followed by a period of retarded growth characterized by the presence of a constant fraction of nonviable cells in the irradiated population. The delayed exposure of cells to caffeine after irradiation showed that the magnitude of the caffeine sensitization diminished throughout the division-delay period. An action spectrum indicated probable nucleoprotein involvement in the induction of division delay. UV light retarded ribonucleic acid and protein synthesis and temporarily blocked deoxyribonucleic acid synthesis. However, synthesis of all three accelerated prior to the end of the division-delay period and then closely paralleled the increase in cell number.     

Inhibition of cell adhesion in Dictyostelium discoideum by tunicamycin is prevented by leupeptin

The carbohydrate requirement for cell adhesion of aggregation-competent cells of Dictyostelium discoideum has been examined by use of a selective glycosylation inhibitor of N-glycosyl protein, tunicamycin (TM). TM completely inhibited EDTA-stable cell adhesion and glycosylation of some membrane glycoproteins in aggregation-competent cells of D. discoideum (Yamada, H., et al. (1982) J. Biochem. 92, 399-406). The present study showed that the inhibition of EDTA-stable cell adhesion by TM was prevented significantly when the cells were treated with TM in the presence of a protease inhibitor, leupeptin (LP), whereas the inhibition of glycosylation by TM was not prevented. The cell extract of aggregation-competent cells contained acid proteases, and LP strongly inhibited acid protease from D. discoideum in vitro. On analysis by SDS-polyacrylamide gel electrophoresis (PAGE), many protein bands present in the membrane fraction of control cells disappeared or decreased on TM treatment of the cells in the absence of LP, however, some of these proteins were restored when the cells were treated with TM in the presence of LP. These results strongly support an idea that EDTA-stable cell adhesion characteristic to aggregation-competent cells is mediated by glycoproteins with asparagine-linked carbohydrate. However, the requirement for the carbohydrate moiety of the glycoprotein in cell adhesion appears to be indirect in that it acts to protect the protein moiety from proteolytic degradation.     

Inhibition of the development of Dictyostelium discoideum by isolated plasma membranes

The life cycle of Dictyostelium discoideum can be divided into two mutually exclusive phases: growth and development. A distinguishing characteristic of the two phases is the absence of intercellular communication during vegative growth, and the many forms of such interaction during development. We have investigated the role of the cell surface membrane during the aggregation and development of this organism. We have asked the question: Are there molecules on the cell surface which are necessary for aggregation, and if so, can they be isolated in a biologically active membrane preparation? Further, when do these molecules appear during normal development, and does the interaction between two neighboring cell surfaces signal the cell or affect their subsequent development in any way? We have been able to isolate a partially purified plasma membrane fraction which is capable of specifically blocking the aggregation of other cells. Additional characterization of this preparation suggests that isolated aggregation phase membranes display a new, or newly exposed, heat-stable component which is capable of interacting with vegetative cells in such a way as to halt development.     

Inhibition of aggregation by light in the cellular slime mold Dictyostelium discoideum

Aggregation of Dictyostelium amoebae is inhibited by light. White light intensities 10² W · cm^-2 cause an inhibition which reaches a saturation at 2 · 10³ W · cm^-2. The action spectrum, based on photon fluence-response curves, shows a major peak around 405 nm and extends through most of the visible spectrum with a secondary maximum at about 530 nm. The action spectrum of the inhibition of aggregation resembles the action spectrum of accumulations of amoebae in light traps and the action spectrum of photodispersal from light traps; it does not resemble the action spectrum of phototaxis in pseudoplasmodia.     

Inhibition of growth and cellular aggregation of Dictyostelium discoideum by steroid compounds.

A study of the effects of steroid hormones on the development of Dictyostelium discoideum has shown that 4 × 10^?5M progesterone, and to a lesser extent dehydro-epiandrosterone, oestradiol and testosterone, inhibit both the growth and aggregation of these amoebae. Pregnenolone is active at lower concentrations (3 × 10^?6M), but at the level of growth only. Progesterone exerts its inhibitory action throughout the aggregation phase. The steroid prevents starved cells from becoming aggregation competent, and inhibits the aggregation of amoebae previously allowed to achieve the competent stage. However, unlike other agents such as dinitrophenol or EDTA, the steroid does not considerably affect cell morphology. Upon addition of progesterone cells become rounded, but after a brief lag they regain their ability to adhere to solid supports and form pseudopods. The fact that the steroids active on Dictyostelium are among the most liposoluble might indicate that their inhibitory action is dependent upon their incorporation into membrane lipids.     

Dependence on Wavelength and Temperature of Growth Inhibition Induced by UV-B Irradiation

Cotyledons excised from dark-grown seedlings of cucumber (Cucumissativus L.) were cultured in vitro at 20C under visible andUV light. Various UV spectra were obtained by using a seriesof cut-off filters that had different transmittance properties.Growth of cotyledons was inhibited by irradiation at wavelengthsshorter than 320 nm and was not affected by irradiation at longerwavelengths. The synthesis of Chl in the cotyledons was stronglyinhibited by irradiation at 280–300 nm and slightly inhibitedby irradiation at 300–320 nm. Inhibition induced by irradiationat 280–300 nm was not affected by temperature, but theextent of inhibition by irradiation at 300–320 nm wasreduced at 25C. This dependence on temperature of the effectof UV light was also observed in the inhibition of photosynthesis.These results indicate that the mode of inhibition by UV lightat 280–300 nm may be different from that induced by UVlight at 300–320 nm and that two or more mechanisms maybe involved in the harmful effects of UV-B radiation. (Received May 10, 1993; Accepted June 15, 1993)     

Inhibition by CIPC of mitosis and development in Dictyostelium discoideum and the isolation of CIPC-resistant mutants

The anti-mitotic herbicide isopropyl N-(3-chlorophenyl) carbamate (CIPC) prevents the growth of amoebae of Dictyostelium discoideum without killing the cells for a period of time equivalent to one generation. During in-hibition, amoebae accumulate in prophase and metaphase of mitosis. After removal of CIPC, they continue through mitosis and then divide. The addition of CIPC to amoebae under starvation conditions prevents aggregation and concomitant cell elongation. The cells, however, do not lose their ability to adhere to a surface, and they remain viable. When CIPC is added to amoebae which have formed streams, it leads to the disintegration of streams into small clusters of cells and to a loss of cell elongation. Post-aggregation stages of development can be inhibited by CIPC at the mound, slug, or Mexican hat stages. Slugs break apart into distinct aggregates. Mutants resistant to CIPC can be obtained easily. Among these mutants, many become temperature sensititive for growth (27°C) or development (27°C or 15.5°C). Others show various abnormalities at the normal temperature (22°C). Most mutants are cross resistant to the microtubule inhibitors nocodazole and thiabendazole, and some are also resistant to CIPC during development.     

Isolation and Characterization of the Putative Photoreceptor for Phototaxis in Amoebae of the Cellular Slime Mold,Dictyostelium discoideum

Amoebae of the cellular slime mold Dictyostelium discoideum (strain AX2) produce a pigment with an absorption spectrum that closely resembles the action spectrum for phototaxis. The protein-pigment complex was isolated and purified by sucrose gradient centrifugation, fast protein liquid chromatography (FPLC) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). It is tightly membrane-bound and the bulk of it is located in the mitochondrial membrane fraction, while a small part is located in the cytoplasmic membrane fraction, as indicated by marker enzyme tests (succinate dehydrogenase for mitochondria and alkaline phosphatase for the cytoplasmic membrane). It is speculated that the pigment bound to the cytoplasmic membrane acts as photoreceptor and that bound to the mitochondria operates as a shading pigment in the light direction perception mechanism of Dictyostelium amoebae.     

Chemiluminescence in Dictyostelium discoideum

Abstract Oxygen radicals generated during oxidative metabolism participate in chemical reactions resulting in light emission. Chemiluminescence is used therefore to measure their production. We have shown that starvation and heat shock induce chemiluminescence in Dictyostelium discoideum . The peak light emission was found to occur about 4 h after the onset of starvation. The optimum temperature for chemiluminescence by starving amoebae was about 33°C. The heat shock inducibility of chemiluminescence was maximal at the beginning of development. Our results are consistent with suggestions that the product(s) of perturbed mitochondrial metabolism might be intracellular signal(s) controlling gene expression in stressed cells. They also suggest a role for intracellular stress signal(s) in the initiation of development in Dictyostelium by starvation.     

Multidirectional phototaxis by Dictyostelium discoideum amoebae

Abstract Phototaxis by solitary Dictyostelium discoideum amoebae is known to be complex, the amoebae turning either towards or away from the light, depending on conditions such as light intensity. Having previously shown that amoebal phototaxis can be bidirectional (2 preferred directions either side of the light source), we now report the discovery of multidirectional phototaxis by D. discoideum amoebae, with up to 12 different preferred directions. As in the bidirectional case, multidirectional phototaxis depends on direction-dependent transitions from turning away from to turning towards the light source.