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.     

Changes in cellular adhesiveness during the development of the slime mold Dictyostelium discoideum

A method was devised to measure the adhesiveness to the substratum of the amoebae of the cellular slime mold, Dictyostelium discoideum, and measurements were conducted with the cells at various stages of development. The adhesiveness of the vegetative amoebae was low, and remained unchanged as long as they fed on bacteria. During the transition from the vegetative stage to the interphase (due to the cessation of feeding), the adhesiveness increased rapidly, and afterwards continued to rise, as development proceeded. The adhesiveness of the interphase amoebae was greatly decreased by the treatment with proteolytic enzymes, lipase, and acid phosphatase. These indicate that accumulation of some substance(s) such as lipoprotein on the cell surface is responsible for the increase in adhesiveness during the interphase. EDTA and periodic acid had no noticeable effect on the adhesiveness of the interphase amoebae. EDTA, however, decreased the adhesiveness in co-operation with trypsin or lipase. The cells disaggregated from the anterior part of the migrating slug showed higher adhesiveness than those from the posterior part. The adhesiveness of either cells was higher than that of the interphase amoebae.     

Alterations in trehalase solubility during development in the cellular slime mould Dictyostelium discoideum

Previous studies have indicated that during development in the slime mould Dictyostelium discoideum, compartmentation of the isoenzymes of trehalase (alpha, alpha'-trehalose 1-D-glucohydrolase, (EC 3.2.1.28) occurs between the extracellular and intracellular environments. The compartmentation of trehalase between soluble and particulate cell fractions was examined in this work. The trehalase present in crude homogenates prepared during the first 12 h of development was completely soluble. Starting at about the pseudoplasmodial stage (i.e. the 14th hour of development), trehalase activity became associated with insoluble cellular material and this increased to a maximal value in homogenates from mature sorocarps, where 50% of the activity was insoluble. Spore cells accounted for only 2 to 3% of the trehalase associated with mature sorocarps, with the remaining 97% being localized in stalk cell material. Although trehalase recovered from spores was completely soluble, more than half of that from the stalk was recovered in the buffer-insoluble pellet fraction.     

Localization of fluorescent substances in the cellular slime moldDictyostelium discoideum cells during growth and development

The localization of fluorescent substance was observed microscopically in livingDictyostelium discoideum cells. The fluorescence was localized in the vacuoles of the vegetative cells. The fluorescent vacuoles were not observed in the dead cells. The fluorescent vacuoles in the cytoplasm were lost in starved cells which are able to form an aggregate and to differentiate. The fluorescent vacuoles were not lost but decreased slightly in the cytoplasm of full grown cells and of cells grown in liquid nutrient medium for an extended period of time (stationary phase cells). On a solid substratum, fluorescent vacuoles were also lost from the cells, where the vegetative cells aggregate and form a slug-shaped mass of cells. The whole slug showed homogeneous fluorescence. In a finally constructed fruiting body, the spore mass showed fluorescence. In a spore mass, the fluorescence was not observed in the spores but in the interspore space of the spore mass. It is suggested that vegetative cells secrete fluorescent substance into the inter-cellular space in the mass of cells during development.     

Cyclic AMP levels and turnover during development of the cellular slime mold Dictyostelium discoideum.

Changes in intracellular and extracellular cAMP levels are reported for the cellular slime mold Dictyostelium discoideum during its development on filter supports. Examined were axenically and bacterially grown strain A3 and bacterially grown NC-4. In each case a major peak in cAMP occurred during aggregation. In addition, axenically grown A3 showed minor rises in cAMP at 16 hr and during culmination; in contrast, NC-4 showed no increase at 16 hr but gave a very large increase at culmination. Both cell-associated phosphodiesterase and the extracellular phosphodiesterase present in the top filter were measured throughout development. Both showed activity peaks during aggregation with much lower plateau values thereafter. At aggregation about 80% of the activity per filter was contributed by the cell-associated phosphodiesterase. The rate of cAMP turnover during aggregation was estimated by following the hydrolysis of applied [³H]cAMP. A minimum rate of about 7% turnover/sec was obtained. From this turnover rate a minimum value for the stimulated activity of the adenylate cyclase was estimated as 224 pmoles/min-mg. Although this level is already over threefold greater than the highest value obtained in vitro, other experiments indicate that the in vivo adenylate cyclase activity may exceed 700 pmoles/min-mg.     

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.     

Inhibition of the development of the cellular slime mould Dictyostelium discoideum by omega-aminocarboxylic acids.

Four omega-aminocarboxylic acids - epsilon-aminocaproic acid (EACA), trans-4-aminomethylcyclohexane-1-carboxylic acid (t-AMCHA), p-aminomethylbenzoic acid (PAMBA) and omega-aminocaprylic acid (OACA) -- prevented fruiting body formation of the cellular slime mould Dictyostelium discoideum. At concentrations of 40 mM, 75 mM, 10 mM and 5 mM, respectively, they allowed aggregation but prevented all further development at 24 degrees C. At lower concentrations, EACA allowed fruiting body formation but with a reduced number of spores per fruiting body. Only t-AMCHA had a significant inhibitory effect on the growth of myxamoebae. EACA affected development only if it was present between 8 and 16 h after the cells were deposited on the filters. Its effect was enhanced by high salt concentrations and by higher temperature, and was also dependent on the manner in which the cells were grown. Only strains capable of axenic growth displayed this sensitivity to EACA, although strains carrying only one of the genetic markers for axenic growth (axe A) were partially sensitive.     

The calcium content of the cellular slime mold, Dictyostelium discoideum, during development and differentiation

A high calcium concentration is known to induce stalk differentiation of the cellular slime mold D. discoideum. Therefore, the change in the calcium content of this organism during differentiation was studied and found to vary during development, more calcium being found in the anterior prestalk cells of the pseudoplasmodium (slug) than in the posterior prespore cells. It is concluded from the results that calcium is of importance in the cell differentiation of this organism and particularly in stalk formation.     

Putrescine uptake by the cellular slime mould dictyostelium discoideum.

1. Rapid labelling occurs when myxamoebae of Dictyostelium discoideum strain AX2 are incubated with [1,4-14C]putrescine. Labelling is energy-dependent. 2. The label enters a pool from which rapid exchange with extracellular putrescine does not occur, and labelling is believed to represent uptake into the cells. 3. The concentration-dependence of putrescine uptake indicates that a number of systems are involved, at least one of which is saturable, with a Km of 9.1 micro M-putrescine. At high putrescine concentrations the overall uptake process is non-saturable. 4. Significant metabolism of putrescine and loss of intracellular putrescine to the medium only occurred when cells were incubated with millimolar concentrations of extracellular putrescine. 5. Putrescine uptake was inhibited by diamines, polyamines, bivalent metal ions and omega-aminocarboxylic acids. 6. The ability to take up putrescine at low concentrations decreased during starvation of myxamoebae. 7. The results are interpreted in terms of a model for putrescine uptake involving adsorptive pinocytosis at low concentrations and fluid-phase pinocytosis at high concentrations.     

Metabolism of halogenated bisphosphonates by the cellular slime mould Dictyostelium discoideum.

Methylenebisphosphonate and its monofluoro-, difluoro- and dichloro- derivatives inhibited growth of amoebae of Dictyostelium discoideum. Dichloromethylenebisphosphonate was the most potent inhibitor of amoebal growth whereas difluoromethylenebisphosphonate was the least potent inhibitor. Each of the bisphosphonates was taken up by the amoebae and incorporated into the corresponding beta, gamma-methylene analogue of adenosine triphosphate. Two of the bisphosphonates were also incorporated into the corresponding analogues of diadenosyl tetraphosphate. No correlation was found between the ability of the bisphosphonates to inhibit amoebal growth and the extent to which they were metabolised.     

Calcium transport in the cellular slime mould Dictyostelium discoideum

Transport of Ca2+ into amoebae of Dictyostelium discoideum was studied using 45Ca and a lanthanum stopping technique. Ca2 uptake was found to be rapid and showed saturation kinetics. No difference was found in Ca2+ uptake between vegetative and aggregation competent cells, the V(max) for unstimulated amoebae being approx. 10 nmol/10(7) cells per min. Ca2+ uptake had the characteristics of passive facilitated diffusion using a saturatable carrier and NaN3 and ouabain were not inhibitory. The chemoattractants cAMP and folate, previously reported to stimulate the uptake of Ca2+ into amoebae, did not stimulate the rate of Ca2+ uptake by this carrier but increased the extent of Ca2+ taken up over the period 10-30 s after chemotactic stimulation. The significance of these findings for the function of Ca2+ in chemotactic signalling is discussed.     

Isozymes of ribonuclease and the changes in their relative levels during development in the cellular slime mould Dictyostelium discoideum

The isozymes of ribonuclease were analyzed in cell-free, crude extracts of Dictyostelium discoideum by activity staining of polyacrylamide gels after electrophoresis. The relative levels of three isozymes were then examined during the growth and during the first stages of multicellular development. We observed the replacement of two of these three isozymes by two other isozymes at the pseudoplasmodial stage. These isozymes were different from ribonuclease T1 in terms of their mobility in polyacrylamide gels during electrophoresis. The mobilities of two of the isozymes, DdI and DdII, were 59 and 42% of that of ribonuclease T1. The changes in the relative levels of the isozymes during development are discussed.     

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.