Germination and mitochondrial damage in spores of Dictyostelium discoideum following supraoptimal heating.

Spores of Dictyostelium discoideum may be quantitatively activated with a heat treatment of 45 degrees C for 30 min. Heat activation at either higher temperatures of for longer duration at 45 degrees C resulted in damaged spores. The spores showed an increased postactivation lag time at 23 degrees C and an increased inability to respond to deactivation with 0.2 M sucrose. As the severity of supraoptimal heating increased, a greater percentage of the spores appeared to contain phase dark lesions and to lose viability. Oxygen uptake began to decrease during and after the appearance of the lesions. Using electron microscopy, the phase dark lesions were found to be mitochondria with disrupted cristae.     

Respiratory competence of Dictyostelium discoideum spores.

Analysis of the respiratory chain of spores of Dictyostelium discoideum, which lack a cyanide-sensitive respiration, indicated that cytochromes a-a3, b, and c-c1 are present at levels identical to those found in the vegetative amoebae. The specific activities of enzymes of both the respiratory chain and the citric acid cycle in the 600 x g supernatant fraction of sonically treated spores were at least as high as in similar preparations of amoebae. The activities of glutamic dehydrogenase and oligomycin-sensitive adenosine triphosphatase were reduced in the spores 30 and 56%, respectively. Intact spores appeared to lack a cyanide-sensitive respiration as a result of inadequate quantities of respiratory substrate and, more importantly, as a result of a lack of the cofactor nicotinamide adenine dinucleotide. The emergence phase of spore germination was sensitive to the antibiotic chloramphenicol, which is a specific inhibitor of mitochondrial protein synthesis. It is concluded that germination requires the early synthesis of oxidized nicotinamide adenine dinucleotide and generation of respiratory substrates and one or more mitochondrially synthesized proteins.     

Gamma-Ray-Induced Spore Germination of Dictyostelium discoideum

⁶⁰Co gamma rays can induce germination of the spores of the cellular slime mold, Dictyostelium discoideum, in the absence of heat shock, amino acids, or bacteria food source. About 65% amoebae emergence occurs by 13 hr after a dose of 180 krad.     

Polyadenylated RNA population present in dormant spores of Dictyostelium discoideum

Total RNA has been isolated from dormant spores of Dictyostelium discoideum. Although the amount of RNA per cell is smaller in spores than in growing amoebae, the ratio of poly(A) sequences to total RNA remains similar. Diversity and base sequence complexity of the polyadenylated RNA population have been examined by molecular hybridization with complementary DNA primed with oligo(dT). By this technique, the number of RNA species detected at more than one copy per cell is approximately 3000. RNA species can be classified in three sets of relative abundance, corresponding respectively to species present on the average at 1000 copies, 50 and four copies per cell. By heterologous hybridization it is shown that a large number of RNA species in spores are the same as those found at other stages of the cell cycle, while 20–30% of the RNA by mass appears specific to the spore cell. The specificity of the spore RNA population resides in the specific accumulation of a small number of RNA species.     

Activation and killing of Dictyostelium discoideum spores with urea.

The optimal conditions for activation of Dictyostellium discoideum spores are an 8 M urea treatment for 30 min. The lag between activation and swelling is 45 min. Lower concentrations of urea do not activate entire spore populations. Incubating spores in 8 M urea for 60 min or treatment with 10 M urea for 30 min results in a lengthening of the post-activation lag and a decrease in the final percentage of germination. Urea-activated spores can be deactivated by azide, cyanide, osmotic pressure, and low-temperature incubation. Activated spores do not germinate if incubated in 1 M urea for 24 h but will complete germination upon resuspension in urea-free buffer. Shocking spores at 45 degrees C in 8 M urea or incubating spores in 4-8 M urea for 10 h at 23.5 degrees C causes inactivation. When suspended in urea-free buffer, a larger percentage of these dead spores release spheroplasts through a longitudinal split in the spore case. Sequential enzyme treatment of spheroplasts with cellulase and pronase causes them to release lysable protoplasts. The data of these experiments suggest that shedding of the outer and middle wall layers during physiological spore swelling may be a physical process rather than an enzymatic one.     

Scanning Electron Microscopy of Spore Germination in Dictyostelium discoideum

The ellipsoidal dormant spores of Dictyostelium dicoideum prepared by freeze-drying had a uniform, compact appearance with fine wrinkles or ridges on the surface. Swollen spores were uneven in appearance, without fine wrinkles but with a seemingly expanded surface covering. The surfaces of the postgermination spore husks appeared unaltered except for a single straight exit slit along the longitudinal plane.     

Ultrastructural Changes During Germination of Dictyostelium discoideum Spores

Spores of Dictyostelium discoideum undergo significant changes in fine structure during germination. The mitochondria progressively become less dense and lose their peripherally attached ribosomes, and the tubuli become more pronounced as germination proceeds. During this period, the three-layered spore wall breaks down in two stages: first, the outer and middle layers are ruptured as a unit, and, second, the inner wall is breached. Crystals and dark (lipid) bodies disappear shortly before or during emergence of the myxamoebae. Autophagic vacuoles are found in dormant spores and throughout the entire germination process. The addition of cycloheximide to germinating spores inhibited the loss of the crystals and dark (lipid) bodies. In addition, the drug inhibited the breakdown of the inner wall layer. Cycloheximide did not prevent the formation of the water expulsion vesicle or the apparent function of the autophagic vacuoles.     

DNA-PKcs-dependent signaling of DNA damage in Dictyostelium discoideum

DNA double-strand breaks (DSBs) can be repaired by either homologous recombination (HR) or nonhomologous end-joining (NHEJ). In vertebrates, the first step in NHEJ is recruitment of the DNA-dependent protein kinase (DNA-PK) to DNA termini. DNA-PK consists of a catalytic subunit (DNA-PKcs) that is recruited to DNA ends by the Ku70/Ku80 heterodimer. Although Ku has been identified in a wide variety of organisms, to date DNA-PKcs has only been identified experimentally in vertebrates. Here, we report the identification of DNA-PK in the nonvertebrate Dictyostelium. Dictyostelium Ku80 contains a conserved domain previously implicated in recruiting DNA-PKcs to DNA and consistent with this observation, we have identified DNA-PKcs in the Dictyostelium genome. Disruption of the gene encoding Dictyostelium DNA-PKcs results in sensitivity to DNA DSBs and defective H2AX phosphorylation in response to this form of DNA damage. However, these phenotypes are only apparent when DNA damage is administered in G(1) phase of the cell cycle. These data illustrate a cell cycle-dependent requirement for Dictyostelium DNA-PK in signaling and combating DNA DSBs and represent the first experimental verification of DNA-PKcs in a nonvertebrate organism.     

Separation of autoinhibitor from cytokinin activity in spores of Dictyostelium discoideum

Spores of Dictyostelium discoideum NC-4H contain a spore germination autoinhibitor (SGI) that, on purification using Sephadex LH-20 eluted with 35% ethanol, can be separated from the bulk of cytokinin activity. Inhibitor studies and chromatographic analysis indicate that SGI is not a known cytokinin such as zeatin or IPA (N⁶-(δ²-isopentenyl)-adenine), N²-dimethylguanosine or discadenine. Considering these findings and conflicting views in the literature concerning the structure of the autoinhibitor, the identity of SGI is discussed.     

Cellulases released during the germination of Dictyostelium discoideum spores.

Dormant spores of Dictyostelium discoideum contained cellulase at a specific activity of 130 to 140 U/mg of protein; when heat activated, the spores germinated, progressively releasing the cellulase activity into the extracellular medium. The cellulase release was a selective process and resulted in recovery of the cellulase activity at a specific activity of 2,000 U/mg of protein; beta-glucosidase in the spores remained completely associated with the emerging amoebae. Release of the cellulase required heat activation of the spores and occurred during the swelling stage of germination; inhibition of the emergence stage with cycloheximide had no effect on the release of the cellulase. The cellulase activity released consisted of two enzymes whose molecular weights were 136,000 and 69,000. Studies of their pH optima, heat lability, and of their sensitivity to inhibition revealed no distinctive differences between these two proteins. Analysis on diethylaminoethyl-Sephadex columns showed that the higher-molecular-weight protein could be converted into the lower-molecular-weight component in vitro.     

Expression of glycosidase activities during germination of Dictyostelium discoideum spores.

Several lysosomal glycosidase activities were examined in vitro during heat-induced germination of Dictyostelium discoideum spores and were found not to be coordinately controlled. The level of beta-glucosidase activity increased significantly during the emergence stage of germination. Both alpha-glucosidase and N-acetyl-beta-glucosaminidase activities remained relatively constant until postemergence, when they increased slightly; alpha-mannosidase activity decreased during all stages of germination. The activity of beta-galactosidase increased slightly during spore swelling, fell below the level initially found in spores at zero time, and increased slightly during postemergence. The expression of all of these enzyme activities, except the increase in beta-galactosidase, appeared to require protein synthesis. Spores in the lag phase of germination which were exposed to severe environmental stress were deactivated and exhibited reduced levels of alpha-glucosidase, beta-glucosidase, and N-acetyl-beta-glucosaminidase activities. Prolonged heat activation treatment reduced the levels of lysosomal glycosidase activities in postactivated spores but did not change the subsequent enzyme patterns during the spore-swelling and emergence stages of germination.     

Factors Affecting the Rate of Heat-induced Spore Germination in Dictyostelium discoideum

Washed spores of Dictyostelium discoideum, strains NC-4H, NC-4D, and V-12, germinated rapidly after being heat shocked at or near 45.0 C for 30 min. Cultures of the slime molds were grown in association with Escherichia coli B/r as the host bacterium; spores taken from plates of synthetic medium had a higher final germination value than spores from complex medium containing peptone and yeast extract. Young spores germinated more rapidly than older spores. Optimal germination occurred between pH 6.0 and 7.0, and, of the buffers tested, potassium phosphate allowed the most rapid germination. After heat shocking, spores were diluted into fresh oxygenated buffer to provide enough oxygen for completion of germination. Germination occurred most rapidly between incubation temperatures of 22 and 25 C.     

S-Adenosyl-L-homocysteine hydrolase is sequestered into actin rods in Dictyostelium discoideum spores.

Here we show evidence that S-adenosyl-L-homocysteine hydrolase (SAHH) is linked to the actin cytoskeleton. Actin rods formed in Dictyostelium discoideum spores during the final stage of development are structurally composed of novel bundles of actin filaments. SAHH only accumulates with actin at this stage of development in the life cycle of D. discoideum. Recently SAHH is believed to be a target for antiviral chemotherapy and the suppression of T cells. Our finding may contribute to designing novel antiviral and immunosuppressive drugs.     

The formation of actin rods composed of actin tubules in Dictyostelium discoideum spores

A new type of actin rod formed in both the nucleus and the cytoplasm, as well as tyrosine phosphorylation of actin, is implicated in the maintenance of dormancy and viability of Dictyostelium discoideum spores. Here the ultrastructure of the rods and their relationship to the phosphorylation of actin were examined. The rods first appeared in premature spores at the midculmination stage as bundles composed of actin tubules hexagonally cross-linked. The 13-nm-diameter bundles were composed of three actin filaments. Formation of the actin rods begins during the late culmination stage and proceeds until 2 days after completion of fruiting bodies. The physical events occur in the following order; association of several modules of bundles, close packing and decrease in diameter of actin tubules, elongation of rods across the nucleus or the cytoplasm. Actin phosphorylation levels increased at the late culmination stage and reached a maximum level 12 h later. Immediately following activation of spore germination, actin was rapidly dephosphorylated, followed shortly thereafter by the disappearance of rods. Shortened actin tubules once again became arranged in a hexagonal pattern. This hexagonal arrangement of actin tubules is possibly involved in rod formation and disappearance and does not depend upon actin phosphorylation. In contrast, rod-maturation processes may correlate with actin phosphorylation.     

Effect of intracellular carbohydrates on heat resistance of Dictyostelium discoideum spores.

The effect of intracellular trehalose and glycogen on the survival of spores of Dictyostelium discoideum ATCC 25697 after exposure to supraoptimal temperatures was examined. Cells metabolically perturbed by incubation in glucose and inorganic phosphate have intracellular trehalose and glycogen concentrations fivefold and twofold higher, respectively, than those of the controls. These cells were more resistant to the lethal effects of wet heat (45 degrees to 55 degrees C) than were control cells. The presence of 40 mM trehalose in the buffer during heat stress increased the survival of nonperturbed cells to approximately the level of the perturbed cells. No protection was observed when cells were heated in the presence of exogenous glycogen. Glucose or disaccharides other than trehalose when present during heat stress, had no effect on heat resistance. Nonperturbed cells preincubated in 40 mM trehalose and washed before heat stress were more resistant to killing than were controls. Cells perturbed with inorganic phosphate, which has been shown to increase trehalose concentrations but decrease glycogen concentrations, were also more resistant to the lethal effects of wet heat than were controls. The data suggest that trehalose has an effect on the wet-heat resistance of D. discoideum. Some possible mechanisms are suggested.     

Tetrahydropteridine deficiency impairs mitochondrial function in Dictyostelium discoideum Ax2

A putative cellular function of tetrahydropteridines (l-erythro-tetrahydrobiopterin and d-threo-tetrahydrobiopterin) was investigated in Dictyostelium discoideum Ax2 using a mutant disrupted in the gene encoding sepiapterin reductase (SR). The SR mutant, which produces about 3% of tetrahydropteridines if compared to wild-type, was elucidated to have several functional defects related to mitochondria and oxidative stress: retarded growth, poor spore viability, impaired mitochondrial function, and increased susceptibility to oxidative stress induced by hydroxylamine or cumene-hydroperoxide. However, the physiological defects were almost completely rescued by extrachromosomal expression of Dictyostelium SR. The results strongly suggested that tetrahydropteridines in Dictyostelium are associated with mitochondrial function, probably via direct protection against oxidative stress.     

Spore Germination in Strains of Dictyostelium discoideum and Other Members of the Dictyosteliaceae

Spores of all strains of Dictyostelium discoideum tested in this study germinated after a heat shock of 45 C for 30 min. Whereas the strains differed in their rates of germination, the rate for each strain was constant. A correlation existed between the rate of germination and the rate of vegetative growth when spores were inoculated into bacterial streaks. Heat shock clearly increased spore germination in D. purpureum, but the response was less dramatic than in D. discoideum. Enhancement also occurred in D. rosarium, but only in media containing peptone. Strains of D. mucoroides gave varied responses, and these could be divided into those which required mutrients for spore germination and those which did not. The spores of Polysphondylium pallidum were resistant to mild heat (45 C), but were not activated; peptone was required for germination. In contrast, the microcysts of this species were heat-labile and required no added nutrients for excystment.     

Mutation frequency of Dictyostelium discoideum spores exposed to the space environment.

Two strains of cellular slime mold Dictyostelium discoideum, a radiosensitive mutant and the parental wild-type strain, were used to investigate the effects of cosmic radiation on viability and mutation frequency at the spore stage for about 9 days in Space Shuttle of NASA. We measured little effect of space environment on viability and cell growth in the both strains as compared to ground controls. The mutation frequency of the flown spores were similar to that of ground control. These results suggest that there could be no effect of cosmic radiation, containing high linear energy transfer radiation at about 0.9 mSv/day as detected by real-time radiation monitoring device on the induction of mutation at the spore stage.