Cytokinin oxidase activity in the cellular slime mold, Dictyostelium discoideum

The cytokinin N6-(delta 2-isopentenyl)adenine (i6Ade) is produced during the development of the cellular slime mold, Dictyostelium discoideum, and functions in this organism as the immediate precursor of the spore germination inhibitor, discadenine. The metabolism of i6Ade in axenic cultures of D. discoideum Ax-3 amoebae has been investigated in the present study. An enzyme activity that specifically catalyzes the degradation of i6Ade has been detected in Ax-3 amoebae. This enzyme is similar to the cytokinin oxidases present in higher plant systems and cleaves the N6-side chain of i6Ade to form adenine. Discadenine synthase activity was also detected in axenically cultured Ax-3 amoebae. The cytokinin oxidase activity detected in Dictyostelium decreased during aggregation and development of Ax-3 amoebae and in starving Ax-3 amoebae maintained under either fast-shake (230 rpm) or slow-shake (70 rpm) conditions. In the latter case, the fall in enzyme activity was accelerated by treatment with cyclic AMP. In contrast to these results, discadenine synthase activity in Ax-3 amoebae rose sharply during the culmination phase of development, exhibited little change in starving Ax-3 amoebae maintained under fast-shake conditions, and fell under slow-shake conditions unless the amoebae were treated with cyclic AMP. Possible functions of the Dictyostelium cytokinin oxidase and the significance of the i6Ade metabolism observed in vegetative Dictyostelium amoebae are discussed.     

Modelling of fluid-phase endocytosis kinetics in the amoebae of the cellular slime mouldDictyostelium discoideum. A multicompartmental approach

Fluid-phase endocytosis (pinocytosis) kinetics were studied inDictyostelium discoideum amoebae from the axenic strain Ax-2 that exhibits high rates of fluid-phase endocytosis when cultured in liquid nutrient media. Fluorescein-labelled dextran (FITC-dextran) was used as a marker in continuous uptake- and in pulse-chase exocytosis experiments. In the latter case, efflux of the marker was monitored on cells loaded for short periods of time and resuspended in marker-free medium. A multicompartmental model was developed which describes satisfactorily fluid-phase endocytosis kinetics. In particular, it accounts correctly for the extended latency period before exocytosis in pulse-chase experiments and it suggests the existence of some sorts of maturation stages in the pathway.     

Metabolism of the cellular slime mould Dictyostelium discoideum grown in axenic culture

1. The DNA, RNA, protein and carbohydrate contents of myxamoebae of Dictyostelium discoideum strain Ax-2 were measured after growth on bacteria or in various axenic media. 2. Myxamoebae grown in the different axenic media have similar DNA, RNA and protein contents, but there are marked differences in the contents of glycogen and free sugars. The DNA and protein contents of myxamoebae grown on bacteria are different from those in myxamoebae grown axenically. 3. Approximately half the DNA found in myxamoebae grown on bacteria is of bacterial rather than of slime-mould origin. 4. The specific activities of some enzymes (including UDP-glucose pyrophosphorylase) are higher in myxamoebae grown axenically than in myxamoebae grown on bacteria. Nevertheless the characteristic increase in the specific activity of UDP-glucose pyrophosphorylase occurring during differentiation of cells of the wild-type strain NC-4 is also found in cells grown axenically. 5. The rate of amino acid oxidation during axenic growth of the myxamoebae is decreased when the cells are supplied with glucose.     

Exploring metal effects and synergistic interactions of ferric stimulation on azo-dye decolorization by new indigenous Acinetobacter guillouiae Ax-9 and Rahnella aquatilis DX2b

The first-attempt study deciphered metal-interacting effects on dye-decolorizing capabilities of indigenous bioelectricity-generating strains, Acinetobacter guillouiae Ax-9 and Rahnella aquatilis DX2b. Most of the metallic ions were inhibitory to color removal capabilities of these strains. However, with supplementation of 5 mM ferric chloride, specific decolorization rate (SDR) of Ax-9 increased by 55.48 % compared to Fe³⁺-free conditions. In contrast, SDR of DX2b decreased 75.35 % due to the inhibition of ferric chloride. On the other hand, ferric citrate could stimulate SDR of DX2b for 21.5 % at same dosage. Enzymatic assay indicated that Fe reductase activity was consistent with synergistic effects of ferric chloride on Ax-9, and ferric citrate on DX2b. Protein analysis via SDS-PAGE and identification of Tandem MS/MS afterwards showed that outer membrane protein (Omp) primarily deals with decolorization as a channeling regulation. Moreover, molecular modeling and bioinformatics data also provided detailed evidences to confirm the biological significance of Omp.     

Specific expression of a gene encoding a novel calcium-binding protein, CAF-1, during transition of Dictyostelium cells from growth to differentiation

The gene expressions involved in the transition from cell proliferation to differentiation were analyzed, using synchronized Dictyostelium discoideum Ax-2 cells and the differential plaque hybridization method. As one of the genes (cDNA) specifically expressed when Ax-2 cells were starved just before the putative shift (PS)-point (putative shift point; a switchover point from growth to differentiation in the cell cycle), calfumirin-1 ( CAF-1 ) was cloned, which encoded a novel calcium-binding protein with E-F hand. Although CAF-1 mRNA was slightly expressed in vegetatively growing cells, the expression was markedly increased in response to starvation of cells just before the PS-point. Northern analysis using non-synchronized Ax-2 cells showed that the CAF-1 mRNA is predominantly expressed within a few hours of starvation. Such a starvation-induced early expression of the CAF-1 mRNA raised a possibility that CAF-1 might be one of Ca²⁺-binding proteins involved in the phase-shift of cells from growth to differentiation.     

Pinocytosis in Dictyostelium discoideum cells : A possible implication of cytoskeletal actin for pinocytotic activity

Pinocytosis in Dictyostelium discoideum axenic strain (Ax-2) cells in the growth phase is progressively inhibited at higher Ca2+ concentrations, the activity being maximal at submicromolar Ca2+ concentrations. The cytoskeletal actin content is also markedly reduced in the presence of 10 mM EGTA. This was confirmed by electronmicroscopy using intact cells and Triton X-100-insoluble cell cortices. Interestingly, the pinocytotic activity seemed to be somewhat increased in response to cytochalasin B (CB). Aggregation-competent Ax-2 cells which are usually devoid of pinocytotic activity can resume their activity considerably following treatment with 10 mM EGTA. Under these conditions, cytoskeletal actin declines markedly, as also was the case for growing Ax-2 cells. Our findings indicate a correlation between the pinocytotic activity and presence of cytoskeletal actin: reduced amounts of actin in the cell cortex seem to favour pinocytosis. Conceivably, membrane-associated actin filaments may function as a powerful anchor, restricting the flexibility of the cell membrane and thereby inhibiting the pinosome formation. Other properties of pinocytosis like a developmental change as well as the effects of pH and temperature are also described and were compared with the properties of wild-type strain, NC-4.     

Transient expression of a mitochondrial gene cluster including rps4 is essential for the phase-shift of Dictyostelium cells from growth to differentiation

Using synchronized Dictyostelium discoideum Ax-2 cells and the differential display method, a mitochondrial gene cluster (referred to as differentiation-associated gene 3; dia3) was isolated as one of the genes expressed specifically during the transition of Ax-2 cells from growth to differentiation. The dia3 gene encodes for a mitochondrial protein cluster (NADH dehydrogenase (NAD) subunit 11, 5, ribosomal protein S4 (RPS4), RPS2, and NAD4L). Northern blot analysis using nonsynchronized Ax-2 cells has shown that the dia3 RNA of about 8 kb is scarcely expressed during the vegetative growth phase, and the maximal expression was attained at 2 h after starvation. To analyze the gene function of dia3, we tried inactivation of rps4 by means of homologous recombination and obtained several transformed clones showing mitochondrial DNA heteroplasmy. The transformed cells grew normally in nutrient medium, but their development after starvation was greatly impaired, thus resulting in the failure of many cells to differentiate. In this connection, the cAMP receptor 1 (car1) expression, which is one of the earliest markers of differentiation, was found to be markedly reduced in the rps4-inactivated cells.     

Relevance of histone H1 kinase activity to the G2/M transition during the cell cycle ofDictyostelium discoideum

The implication of histone H1 kinase activity for the G2/M transition during the cell cycle was investigated usingDictyostelium discoideum Ax-2. Histone H1 kinase with its activity was purified from cell extracts by the use of p13^suc1 affinity gel. In the vegetative cell cycle, the activity of histone H1 kinase including Cdc2 kinase was found using synchronized Ax-2 cells to be highest just before the entry into mitosis. The activity also was markedly enhanced just prior to the M phase from which developing cells (possibly prespore cells) reinitiate their cell cycle at the mound-tipped aggregate stage. These results strongly suggest the importance of Cdc2 kinase activity in the G2 to M phase transition during the cell cycle, as the case for other eukaryotic cells.     

Cytofluorimetric research on the changes in the DNA content in the nuclei of Amoeba proteus during prolonged starvation and after refeeding]

Dividing amoebae were manually selected from the culture of Amoeba proteus, and so groups of synchronously dividing (synchronized) amoebae were obtained. These synchronized amoebae were maintained without food. In spite of starvation, individual amoebae in some particular groups were seen to divide, whereas in other groups of amoebae there was no division at all. The starving amoebae died not earlier than 2 weeks after the last division. A relative DNA content in isolated nuclei has been determined cytofluorometrically for each of 6 groups of synchronized starving amoebae, unable to divide. The nuclei were isolated in different intervals after division (after the feeding was ceased): 1.0-1.5 h, 1 day and up to 13 days with 1-2 day intervals. In the all groups of amoebae DNA synthesis occurred on the first 1-2 days after division. The nuclear DNA content in amoebae of 3 groups increased more than two-fold as compared with the 1 h level, in other 3 groups the nuclear DNA content did not exceed the doubled 1 h level, but probably exceeded the doubled postmitotic level. Later on, the nuclear DNA content in starving amoebae of each group was seen to decrease by 16-20%. Amoebae of 3 of the 6 groups were given the food organisms (Tetrahymena pyriformis) 8 days after division (after cessation of feeding). 2-3 days after refeeding some of these amoebae divided, and the nuclear DNA content of the refed amoebae proved to be higher than that in amoebae that continued to starve. It is suggested that the decrease of DNA content in the nuclei of starving amoebae and the increase of DNA quantity in the nuclei of refed amoebae may result from degradation and induction of synthesis of specific extra DNA synthesized in amoeba nuclei during each cell cycle.     

Transition of starving Dictyostelium cells to differentiation phase at a particular position of the cell cycle

The relationship between proliferation and differentiation in Dictyostelium discoideum Ax-2 was analyzed with reference to the cell-cycle position at the onset of starvation, using cells synchronized by temperature shift (11.5 degrees C-22.0 degrees C). To examine how far Ax-2 cells at any particular phase of the cell cycle are able to progress through the cycle in response to nutritional deprivation, we measured temporal changes in cell number and nuclearity after starvation. Nuclear DNA synthesis in synchronously developing cells was also monitored by pulse-labeling with [methyl-3H]thymidine. Increase in cell number and subsequent DNA synthesis occurred in cells just before mitosis (referred to as T0.5 cells and T1 cells; 0.5 h and 1 h after the shift-up from 11.5 degrees C to 22.0 degrees C respectively), but not in T3, T5, or T7 cells. When T1 cells were incubated for 6 h in the absence of external nutrients, they (T1 + 6 cells) exhibited developmental features similar to T7 cells, which most rapidly acquired chemotactic sensitivity to 3',5'-cyclic adenosine monophosphate (cAMP) and EDTA-resistant cohesiveness after starvation. Thus, it is quite likely that Ax-2 cells may progress through the cell cycle to a particular point (possibly the cell-cycle position of T7 cells), irrespective of the presence or absence of nutrients, and enter the differentiation phase from this point under conditions of nutritional deprivation. There was no difference in the ratio of prestalk to prespore cells in migratory pseudoplasmodia derived from cells that had been starved at other cell-cycle positions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclic GMP and cyclic AMP changes in response to folic acid pulses during cell development of Dictyostelium discoideum.

Folic acid pulses induced developmental processes in agip 71, a morphogenetic mutant of Dictyostelium discoideum, strain Ax-2. Cells that had received folic acid pulses were able to form EDTA-stable cell aggregates and to complete full differentiation to fruiting bodies. In these cells no autonomous periodic activities were observed by light scattering. Folic acid pulses elicited increases in the concentrations of cyclic GMP and cyclic AMP. In undifferentiated cells, folic acid caused a rapid increase in the level of cyclic GMP without a significant change in the level of cyclic AMP. In an advanced developmental state folic acid caused an increase in cyclic AMP in addition to two successsive peaks of cyclic GMP. Experiments performed with the parent strain, Ax-2, also showed that during the development towards aggregation competence, cells acquired the ability to produce a cyclic AMP peak in response to folic acid.     

Possible involvements of 101 kDa, 90 kDa, and 32 kDa phosphoproteins in the phase-shift of Dictyostelium cells from growth to differentiation

Abstract. As demonstrated previously, the transition of starving Dictyostelium cells from growth to differentiation phase occurs at a particular position (putative shift point; PS-point) in G₂-phase of the cell cycle of Dictyostelium discoideum Ax-2. In this study we examined what proteins are phosphorylated or dephosphorylated at the onset of starvation, with special emphasis on changes of phosphoproteins near the PS-point. When AX-2 cells at any particular phase of the cell cycle were pulse-labeled with inorganic ³²P (³²P_i) in the presence or absence of nutrients, it was found that 101 kDa and 90 kDa phosphoproteins exhibit specific changes around the PS-point. From the chase-experiments of ³²P-labeled cells, the 101 kDa and 90 kDa proteins were found to fail to be phosphorylated at the PS-point under starvation conditions. The protein phosphatase inhibitors such as okadaic acid and calyculin A inhibited completely entry of starving Ax-2 cells to differentiation, and also blocked perfectly dephosphorylation of 32 kDa protein. Taken together it is likely that dephosphorylation of 32 kDa protein as well as low phosphorylation levels of 101 kDa and 90 kDa proteins may be required for the phase-shift of Ax-2 cells from growth to differentiation. Subcellular fractionation showed the 101 kDa phosphoprotein to be located in cytoplasm, while parts, at least, of the 90 kDa and 32 kDa phosproproteins were in the nucleus. In addition, the results of cellulose thin-layer electrophoresis of digested 101 kDa and 90 kDa phosphoproteins show that in both proteins only serine residues are phosphorylated. The significance of phosphorylation states of 101 kDa, 90 kDa, and 32 kDa proteins is discussed in relation to a breakaway of cells from proliferation to differentiation.     

Involvement of the glucose-regulated protein 94 (Dd-GRP94) in starvation response of Dictyostelium discoideum cells

Upon deprivation of nutrients, Dictyostelium discoideum Ax-2 cells arrest proliferation and initiate a metamorphosed developmental program including induction of altered gene expressions which are necessary for differentiation. In Ax-2 cells, we found out a member of Hsp90 family usually contained in the endoplasmic reticulum (ER), Dd-GRP94 (Dictyostelium discoideum glucose-regulated protein 94). In general, GRP94 are induced either by glucose-depletion or by depletion of Ca(2+) in intracellular Ca(2+) stores. Unexpectedly, however, the expression of Dd-grp94 was greatly reduced within 60 min of starvation. Dd-grp94-overexpressing cells (GRP94(OE) cells) collected without forming distinct aggregation streams, and never formed normal fruiting bodies. Also, prespore differentiation as well as maturation into spores and stalk cells were particularly impaired in the GRP94(OE) cells. Thus Dd-GRP94 seems to be crucial in late differentiation as well as in starvation response.     

Abundance of Naked Amoebae in Sediments of Hiroshima Bay, Seto Inland Sea of Japan

ABSTRACT The present paper provides the first data on naked amoebae from sediments of Hiroshima Bay. Three stations in the inner part of the bay were sampled over a three-month period. Abundance of naked amoebae ranged from 1,019 to 45,561 cells/g dry sediment. Results indicate: (i) surface sediment populations in most cases were higher than subsurface populations; (ii) there was some evidence of temporal variation with counts generally increasing from March to May: and (iii) the site located near Hiroshima City had fewer amoebae on several occasions than the other two sites. There was a negative exponential relationship between acid-volatile sulfide concentration and abundance of amoebae. Most amoebae were small with the average size ranging from 6.6–14 μm. Morphotype 1, amoebae that extend lobose pseudopodia or subpseudopodia during normal locomotion, were dominant (40–100% of enumerated amoebae). Morphotypes 2 and 3 (limax amoebae) were found in lower numbers than the other two morphotypes. The proportion of amoebae occupied by Morphotype 4 (fan-shaped or discoidal-flattened amoebae) was higher at a lower total abundance.     

Concanavalin A-mediated agglutination and distribution of concanavalin A-binding sites in Acanthamoeba following treatment with colchicine and cytochalasin B

Incubation of Acanthamoeba castellanii (Neff strain) with FITC-ConA (15 micrograms/ml) resulted in the appearance of patches of fluorescence on the amoebae within 2 min of incubation. These patches disappeared following treatment of the amoebae with alpha-MeMan. Pretreatment of the amoebae with colchicine or cytochalasin B or with colchicine and cytochalasin B in combination did not significantly alter the distribution pattern of fluorescence in the amoebae. 2,4-Dinitrophenol and incubation at 4 degrees C on the other hand decreased the degree of patching of the amoebae. Pretreatment with 2,4-dinitrophenol and incubation at 4 degrees C also decreased the ConA-mediated agglutination of the amoebae. No effect on the ConA-mediated agglutination was, however, observed following pretreatment of the amoebae with colchicine and cytochalasin B neither alone nor in combination. Our results indicate that ConA-mediated agglutination and long-range ConA-receptor mobility in the Acanthamoeba are not under the control of structures sensitive to cytochalasin B or colchicine.     

Adenosine 3′:5′-cyclic monophosphate-binding protein from the cellular slime mould Dictyostelium discoideum (Short Communication)

During the differentiation of myxamoebae of Dictyostelium discoideum strain Ax-2 grown in axenic medium there is a seven- to ten-fold increase in the specific activity of cyclic AMP-binding protein(s). Evidence is presented for the belief that cyclic AMP phosphodiesterase and cyclic AMP-binding protein are distinct molecular species.     

Changes of Endocytotic Activities during the Cell Cycle of Dictyostelium Cells

Changes of endocytotic activities during the cell cycle of the cellular slime mould Dictyostelium discoideum Ax-2 were examined using the temperature-shift method for inducing synchronous growth. The activity of fluid-phase pinocytosis (FPP) was altered Ca²⁺-dependently and stimulated by EGTA. On the other hand, pinocytosis was greatly enhanced by addition of Bacteriological-peptone(BP) to the growth medium for Ax-2 cells, irrespective of the extracellular Ca²⁺-concentration. The maximal pinocytotic activity was attained in the presence of EGTA plus BP, the effects of the two substances being additive. The FPP activity was found to be high in cells in and just after the S phase, when the BP-induced fraction of pinocytosis was rather low. Thus the total activity for pinocytosis in the growth medium remained almost constant throughout the cell cycle, indicating that the rate of nutrient uptake through pinocytosis was not a limiting factor for cell cycle regulation. The change of phagocytotic activity during the cell cycle was somewhat similar to that of the FPP activity. Possible mechanisms of such cell-cycle related changes are discussed in relation to cytoskeletal proteins in the cell cortex. Some properties of BP-induced pinocytosis are also described.     

Resumption of locomotion byAmoeba proteus readhering to different substrata

Summary Floating heterotactic cells ofAmoeba proteus were sedimented on untreated glass surfaces and on modified substrata, differing in their wettability and surface potential. About 95% of the amoebae readhere to the glass within 12 min and recover locomotive (polytactic) morphology within 13 min. The rate of locomotion resumption does not change significantly on styrene/methyl methacrylate co-polymers with contrasting hydrophilic sulfonic group surface densities. Almost all amoebae readhere within 3 min to the positively charged surface of polylysine-coated glass, but locomotive shape is only reassumed after 20 min by 95% of them. The polytactic cells are marked flattened on polylysine and move 2 1/2 times more slowly than on the glass. Floating amoebae never readhere to negatively charged gelatin gel; up to 25% become polytactic after 20 min, but they never resume locomotion. Indifference of amoebae to substratum wettability, and their prompt reaction to the positively or negatively charged surfaces, are discussed. The polylysine and gelatin gel substrata seem suitable for the study of adhesion dependent motor functions in amoebae.     

Testate amoebae taxonomy and trait diversity are coupled along an openness and wetness gradient in pine-dominated Baltic bogs

Sphagnum peatlands host a high abundance of protists, especially testate amoebae. Here, we designed a study to investigate the functional diversity of testate amoebae in relation to wetness and forest cover in Baltic bogs. We provided new data on the influence of openness/wetness gradient on testate amoebae communities, showing significant differences in selected testate amoebae (TA) traits. Three key messages emerged from our investigations: 1) we recorded an effect of peatland surface openness on testate amoebae functional traits that led us to accept the hypothesis that TA traits differ according to light intensity and hydrology. Mixotrophic species were recorded in high relative abundance in open plots, whereas they were nearly absent in forested sites; 2) we revealed a hydrological threshold for the occurrence of mixotrophic testate amoebae that might be very important in terms of peatland functioning and carbon sink vs. source context; and 3) mixotrophic species with organic tests were nearly absent in forested sites that were dominated by heterotrophic species with agglutinated or idiosomic tests. An important message from this study is that taxonomy of TA rather indicates the hydrological gradient whereas traits of mixotrophs the openness gradient.     

Characterization of DNA from the cellular slime mould Dictyostelium discoideum after growth of the amoebae in different media

Amoebae of the cellular slime mould Dictyostelium discoideum Ax2 grown on Aerobacter aerogenes as food source have a DNA content (36.0 ± 0.9 × 10⁻¹⁴ g/cell) approximately twice that of the same amoebae grown axenically (16.8 ± 0.4 × 10⁻¹⁴ g/cell). Isolation and characterization of DNA from amoebae grown either axenically or on bacteria, by several methods (melting curve, density gradient centrifugation, DNA/DNA hybridization) suggests that not more than 16% of the DNA content of bacterially grown amoebae is of bacterial origin. Studies of the rate of reannealing of DNA samples isolated from amoebae grown either axenically or on bacteria and of the degree to which they hybridize with ribosomal RNA, suggests that the ‘extra’ DNA that bacterially grown cells contain is biologically similar to that contained in axenically grown cells. It is therefore concluded that amoebae growing exponentially on bacteria have, on average, 2.4 to 2.7 genome equivalents per cell and amoebae growing exponentially in axenic medium have 1.3 to 1.4 genome equivalents per cell. Since it is believed that amoebae of this strain growing on bacteria are haploid and since these differences in DNA content persist during their subsequent differentiation, it is concluded that axenically grown amoebae differentiate whilst in the G1 phase of the cell cycle and bacterially grown amoebae differentiate whilst in the G2 phase of the cell cycle.