Aggregateless Mutants Research Articles

CAMP stimulation of Dictyostelium discoideum destabilizes the mRNA for 117 antigen.

Transcription of the 117 gene and changes in its mRNA levels in Dictyostelium discoideum were studied by mRNA hybridization with a cDNA probe. In wild type cells (Ax-2), the expression is developmentally regulated during cell aggregation, while in the aggregateless mutant, Agip 45, 117 mRNA is not detectable during cell starvation. Low concentrations of cAMP, given in the form of extracellular pulses to induce the development of starved Agip 45 cells to aggregation competence, are able to induce the appearance of 117 mRNA. The induction seems to be via the cell surface cAMP receptor and by a mechanism which does not involve changes in intracellular cAMP. Interestingly, high concentrations of cAMP, which down-regulate the cell surface cAMP receptor, elicit a rapid decrease in the level of 117 mRNA in aggregation-competent cells. Nuclear run-off and pulse-chase experiments show that the high concentrations of cAMP selectively destabilize the mRNA for 117 antigen. This destabilization requires both de novo mRNA synthesis and protein synthesis since the addition of inhibitors of these processes eliminates the effects of cAMP on 117 mRNA. The data suggest that a cAMP-induced protein(s) may be involved in the destabilization of selective mRNAs.

Founder Cell Differentiation and Acrasin Production in an Aggregateless Mutant of Polyspondylium violaceum: (aggregation/D factor/founder cell/glorin/Polysphondylium violaceum).

A specific class of aggregation-deficient mutants, aggA, of Polyshondylium violaceum are unable to aggregate unless supplied exogenously with a stimulating factor called D factor. The present study examines the effect of D factor on the induction of founder cells and on the production of the chemoattractant of aggregation, N-propionyl-γ-L-glutamyl-L-ornithine-δ-lactam ethyl ester (or glorin). Founder cells initiate aggregate formation and are morphologically distinct from the majority of the amoebae. Founder cell differentiation and oriented movement of attracted amoebae have been studied by time-lapse videotape analysis. In wild-type strains, on the average 90 min after the onset of starvation, a single, motile, irregularly shaped amoeba stops wandering and becomes round in shape. This founder cell has differentiated randomly from the pool of starved amoebae and within 2.5 min after the cessation of movement begins to attract and establish cellular contacts nighboring amoebae. The aggA mutants neither aggregate nor differentiate founder cells in the absence of D factor; whereas, aggregate formation and founder cell differentiation occur in the presence of physiological concentrations of purified, externally added D factor. However, in either the presence or absence of D factor, aggA amoebae produce and excrete glorin (measured using a bioassay) at levels comparable to their parental strain. These studies suggest that D factor is required for founder cell differentiation and organization of the aggregate, and that the ability to synthesize and excrete glorin is not sufficient to trigger aggregation.

Analysis of developmentally defective chemical signaling mutants of Polysphondylium violaceum.

Six aggregation-defective mutants of Polysphondylium violaceum dependent on external addition of the pheromone D factor for aggregation were isolated after nitrosoguanidine mutagenesis. With a screening technique based on synergistic development, D-factor-dependent mutants can be separated from other kinds of aggregateless mutants. Genetic complementation analyses of the newly isolated mutants showed them to be mutant at the aggA locus. Individual mutants exhibited different sensitivities to D factor(s), responding maximally over a 300-fold range of concentrations.

Developmental regulation of production of an aggregation-stimulating factor from the cellular slime mold Polysphondylirrm violaceurn

An excreted, dialyzable component(s) produced during development of wild-type Polysphondylium violaceum has been previously shown to stimulate aggregation of aggregateless mutants in the complementation group agg A. Production of this aggregation-stimulating factor, called D factor, is greater during development in liquid culture than during development on a surface. After partial purification of crude D factor using high performance liquid chromatography, multiple species are found that retain the ability to stimulate aggregation of the agg A mutants. The three major components (Df A, Df B, and Df C), show decreasing polarity based on purification using reverse-phase chromatography. The proportion of each component secreted varies, depending on the developmental conditions (surface versus liquid) and the time after starvation when the factors are isolated. Preliminary physical and chemical characterization of the three D factor components suggests that they are related.

Vesicle-mediated transfer of phospholipids to plasma membrane during cell aggregation of Dictyostelium discoideum.

We have previously reported that synthesis of phospholipids increases 4-fold at the onset of chemotactic migration during development of Dictyostelium discoideum and that the newly synthesized phospholipids are preferentially incorporated into the plasma membrane (De Silva, N. S. and Siu, C-H. (1980) J. Biol. Chem. 255, 8489-8496). To test the hypothesis that the rapid transfer of phospholipids to the plasma membrane is mediated by vesicles, we isolated phospholipid-rich vesicles from cells at 6 h of development. These vesicles had an average size of 0.35 micrometer in diameter. They banded at a density of 1.097 g/cm3 and they had a phospholipid: protein (w/w) ratio of 2.25. The predominant classes of phospholipids in these vesicles were phosphatidylethanolamine and phosphatidylcholine. In pulse-labeling studies using [3H]glycerol, these low density vesicles had the highest phospholipid-specific activity, which was about 3 times higher than that of 6-h plasma membranes. Almost 80% of the incorporated radioactivity was found to be associated with phosphatidylethanolamine and phosphatidylcholine. When cells were chased with cold precursor after pulse labeling, the specific activity of these vesicles dropped by almost 20-fold in 90 min, while plasma membranes showed a 2.5-fold increase in 60 min. Addition of colchicine to 7-h cells inhibited the translocation of newly synthesized phospholipids to the plasma membrane. The low density vesicles were found in much reduced amounts in preaggregation stage cells or the aggregateless mutant WL3. These results indicate that transfer of newly synthesized phospholipids from their site of synthesis to the plasma membrane probably occurs through a special class of phospholipid-rich vesicles.

Synthesis of stage-specific glycoproteins in Dictyostelium discoideum during development

Synthesis of glycoproteins by the cellular slime mold Dictyostelium discoideum during development was monitored by the incorporation of [ 3 H]fucose. The patterns of glycoproteins synthesized at the aggregations tage and the culmination stage showed marked differences. When cells were actively undergoing chemotactic migration and cell streaming, at least five clearly identifiable glycoproteins were synthesized, with apparent molecular weights of 165,000, 150,000, 125,000, 108,000, and 80,000. Synthesis of these glycoproteins was greatly reduced after 10 hr and completely terminated by 12 hr. Also their synthesis was blocked in aggregateless mutants. An approximate eightfold increase in the rate of [ 3 H]fucose incorporation into glycoproteins was observed between 17 and 21 hr, corresponding to the onset of the culmination stage. Three major glycoproteins with apparent molecular weights of 103,000 (gp103), 80,000, and 72,000 appeared to accumulate in cells during this period. They were synthesized predominantly in the prespore cells and were negligible in prestalk cells, which had a much lower incorporation of [ 3 H]fucose. Many of the prespore-specific glycoproteins were preserved in the mature spores and became associated with the spore coat. gp103 was found to accumulate first inside the cell during culmination and translocated later to the cell surface to become part of the spore coat.

AGGREGATELESS MUTANT DEFECTIVE IN SIGNALING AND RELAYING IN THE CELLULAR SLIME MOLD, DICTYOSTELIUM DISCOIDEUM.

An aggregateless mutant (HT41), isolated from D. discoideum NP14, was examined for the parameters involved in cell aggregation. HT41 cells were induced to express some of these parameters by cyclic AMP pulses. Spontaneous oscillations in light scattering were not observed in the suspension of the mutant cells. A pulse of cyclic AMP caused a monophasic decrease in light scattering, which was similar to the response of pre-aggregation cells of the wild-type. When cyclic AMP pulses were applied on a cell layer, HT41 cells aggregated without forming the streams. These results suggest that HT41 is a mutant defective in signal emission and relay.

Effect of cyclic AMP pulses on the synthesis of plasma membrane proteins in aggregateless mutants of Dictyostelium discoideum

Effect of cyclic AMP pulses on the synthesis of plasma membrane proteins in aggregateless mutants of Dictyostelium discoideum

Cell contact-induced inhibition of division in Dictyostelium *

The developmental stage at which Dictyostelium discoideum strain v12/M2 cells was inhibited in resuming cell division was monitored by a Coulter counter. Neither vegetative nor pre-aggregating v12/M2 amoebae used as inocula in liquid growth cultures exhibited a delay in recommencing cell division. Inocula prepared from aggregates were delayed for 3 h before the onset of division. Although cyclic AMP had no effect upon vegetative or preaggregating stages the aggregating amoebae were inhibited for 9 h before resuming division. Following the onset of division by both controls and cyclic AMP-treated cells the normal growth rate of vegetative amoebae (3.2 h/generation) was attained. Aggregateless mutant vegetative amoebae and those of comparable ages to vas/M2 aggregates were not inhibited in the rate of cell division by cyclic AMP. Cyclic AMP sustains the delay in cell division only among amoebae derived from aggregates. Cyclic AMP induces structural components in the plasma membranes which may be necessary in cell adhesion and interactions. Consequently interactions between the membranes of apposing cells may transmit stimuli intracellularly to inhibit cell division.

A biochemical study of the effects of cAMP pulses on aggregateless mutants of Dictyostelium discoideum

We have examined the ability of six aggregateless mutants to express the biochemical parameters involved in the production of the chemotactic signal, cAMP pulses. The mutants are representative of the three groups which have been defined previously in terms of the ability of applied cAMP pulses to induce all, or part, of the developmental cycle of nonaggregating strains [ Darmon, M., Brachet, P., and Pereira da Silva, L. (1975). Proc. Nat. Acad. Sci. USA 72, 3163–3166 ; Darmon et al., 1977 ]. Strains were monitored for adenylate cyclase, cellular and extracellular phosphodiesterase activities, the levels of cAMP binding sites, and the extracellular phosphodiesterase inhibitor. The ability of applied cAMP pulses to alter any of these parameters was also examined. The results indicate that adenylate cyclase can exist in active and inactive forms and that applied cAMP pulses effect the transition of the enzyme to its active state. Applied cAMP pulses are also shown to induce the adenylate cyclase activity of two strains and inhibit the production of the phosphodiesterase inhibitor. The data are discussed in terms of the possible sequence of events involved in the differentiation of cells to aggregation competence.

Cell Responsiveness to cAMP during the Aggregation Phase of Dictyostelium discoideum: Comparison between the Inhibitory Action of Progesterone and the Stimulatory Action of EDTA and Ionophore A23187

Amoebae of the cellular slime mould Dictyostelium discoideum undertake a developmental program under conditions of food deprivation. After a lag period of a few hours, called the interphase, differentiated amoebae express the capacity to migrate toward central collection points where they form multicellular aggregates able to generate fruiting bodies. Aggregation-competent amoebae communicate positional information via cAMP pulses. Oscillating levels of cAMP play a dual role in the differentiation of the cells, since they are also responsible for the induction of several cellular properties characteristic of aggregation competence: chemotactic responsiveness, cell cohesiveness and high levels of cAMP binding sites on the plasma membrane. In previous studies, conditions were defined under which the differentiation of amoebae into aggregation-competent cells was either inhibited or stimulated. 4 × 10 −5 M progesterone was shown to prevent aggregation, while 10 −3 M EDTA plus 5 × 10 −3 M MgCl 2 , or 7 × 10 −7 M ionophore A23187 appeared to accelerate this process by shortening the interphase period. In the present work, experiments were carried out to investigate the molecular level at which these compounds exert their action. Progesterone is shown to inhibit aggregation by rendering amoebae unresponsive to the chemotactic signal, cAMP pulses. Even after prolonged periods of starvation, steroid-treated cells do not develop aggregation competence, but retain the properties characteristic of growth phase cells: they fail to respond to cAMP in the chemotactic tests, but remain sensitive to folic acid; they fail to develop EDTA-resistant contacts and cAMP binding sites on their plasma membrane. Steroid-treated amoebae starved in the presence of exogenously generated cAMP pulses cannot be induced to express any of the characteristics of differentiated cells. The stimulation of cell aggregation obtained by starving cells in the presence of EDTA, MgCl 2 or ionophore A23187, is accompanied by an early development of the properties characteristic of aggregation-competent amoebae: increase of the chemotactic sensitivity to cAMP, loss of the sensitivity to folic acid and increase in the number of cAMP binding sites. Although externally applied cAMP pulses do not act cooperatively with these compounds in stimulating cell differentiation, a synergistic effect is observed with a differentiation stimulating factor (DSF), which is known to make amoebae precociously sensitive to cAMP pulses. Incubation of two aggregateless mutants with EDTA, MgCl 2 , or ionophore, phenotypically suppresses their morphogenetic block. The differentiation of these strains has previously been shown to be fully induced by imposed cAMP pulses. The data are interpreted on the basis of a stimulation of adenyl cyclase by EDTA and ionophore A23187, and further emphasize the importance of this enzyme in the differentiation of Dictyostelium cells.

Phenotypic suppression of morphogenetic mutants of Dictyostelium discoideum

The effects of cAMP pulses on the capacity of 15 aggregateless mutants to differentiate and construct fruiting bodies are compared to those obtained when mutant cells are starved with wild-type amoebae. Mutant strains are classified into three main groups depending upon the degree to which their phenotypic defects can be corrected. These data extend studies published earlier [Darmon, M., Brachet, P., and Pereira da Silva, L. (1975). Chemotactic signals induce cell differentiation in Dictyostelium discoideum. Proc. Nat. Acad. Sci. USA 72, 3163–3166; Pereira da Silva, L., Darmon, M., Brachet, P., Klein, C., and Barrand, P. (1975). Induction of cell differentiation by the chemotactic signal in Dictyostelium discoideum. In “Proceedings of the Tenth FEBS Meeting,” pp. 269–276]. (1) Only one mutant was unresponsive both to cAMP pulses and to the presence of wild-type amoebae and did not display any of the properties of differentiated cells. (2) Following treatment with cAMP pulses, 11 mutants developed certain properties of aggregation-competent amoebae. They increased their levels of cellular phosphodiesterase, showed an enhanced chemotactic sensitivity to cAMP, and established specific cell contacts. None of these amoebae could differentiate further. They did co-aggregate to some extent with wild-type cells, but failed to differentiate into spores. Rather, mutant cells were excluded from the pseudoplasmodium during the process of morphogenesis of the fruiting body. (3) In contrast, the aggregateless phenotype of three mutants was fully corrected by both cAMP pulses and the presence of wild-type cells. These findings are discussed on the basis of a relationship between the chemotactic signal and cell differentiation.

Dictyostelium aggregate-less mutant plasma membranes

This investigation concerns a freeze-fracture study of the plasma membranes of aggregate-less mutants 67 and 20-2 derived from the wild-type slime mold Dictyostelium discoideum V12 M2 . These mutants cannot respond chemotactically to cAMP and consequently are incapable of normal fruiting body formation. Freeze-fracture studies of Agg 67 and 20-2 amoebae revealed that the average diameters of the plasma membrane particles were almost identical to the wild-type strain V12 M2 vegetative amoebae. Although exposure to cAMP effected a 1.4 × increase in average particle size in the V12 M2 amoebae membranes, those in the mutant cell types did not increase in average diameters. The state of the plasma membrane and its regulatory role in the cell cycle and cell differentiation is discussed.

Induction of fruiting in two aggregateless mutants of Dictyostelium discoideum

Five general groups of morphogenetically aberrant mutants of Dictyostelium discoideum were isolated. Each group of mutants was characterized either by the absence of any fruiting structures or by the formation of abnormal fructifications. Among these developmental mutants were two aggregateless isolates, Agg −-1 and Agg −-2, that could be induced to form normal sorocarps under certain conditions. Sorocarps of the normal D. discoideum type were formed when growing myxamoebae from either of these mutants were allowed to come in contact with myxamoebae of the other mutants, wild-type D. discoideum, D. purpureum, or D. mucoroides. No sorocarps were formed when myxamoebae of Agg −-1 and Agg −-2 were paired. These two aggregateless mutants, while incapable of aggregating or fruiting when cultivated singly with Escherichia coli B/r on a glucose-salts medium, formed normal fruiting structures after being freed of what appeared to be a product of bacterial growth. The spores produced by Agg −-1 and Agg −-2 myxamoebae again gave rise to aggregateless clones of the original parental types.

Appearance of Wild Type from Mixed Culture of Two Aggregateless Mutants in the Cellular Slime Mold <i>Dictyostelium discodieum</i>

Appearance of Wild Type from Mixed Culture of Two Aggregateless Mutants in the Cellular Slime Mold <i>Dictyostelium discodieum</i>

Some aspects of cell interaction in the development of the slime mold Dictyostelium purpureum

Some aspects of cellular interaction in the slime mold Dictyostelium purpureum Olive have been described, with emphasis on experiments involving an aggregateless mutant and the wild type. The results suggest the following conclusions: 1. 1. In mixed populations with wild-type cells, agg-1 myxamoebae are incorporated into the developing pseudoplasmodia. 2. 2. The presence of agg-1 cells in such mixed populations increases the number of centers of aggregation. The exact nature of this effect is unknown, but an explanation is presented based on the concept of “aggregation territories” ( Bonner and Dodd, 1962). 3. 3. Single wild-type cells can initiate center formation leading to subsequent fruiting in a population of agg-1 myxamoebae, and it is suggested that in wild-type populations any cell may initiate a center. 4. 4. When spores are cloned from sorocarps derived from a mixture of aggregateless and wild-type cells, very few if any plaques of the agg-1 type are recovered. In the absence of suitable alternatives, it is suggested that this apparent loss is due to a “conversion” or “reversion” of agg-1 cells into those of the wild type. Several mechanisms (involving enzyme induction, epigenetic homeostasis or cytoplasmic inheritance) by which this change might occur are examined.

Fatty Acid and Phospholipid Composition of the Cellular Slime Mold, Dictyostelium discoideum: THE OCCURRENCE OF PREVIOUSLY UNDESCRIBED FATTY ACIDS

The cellular slime mold Dictyostelium dticoideum has been extensively studied (1) because of the unique series of morphological transitions that the wild type undergoes. From single, vegetative cells, the organisms pass through complex aggregation, migration, and culmination, leadii finally to spore formation. Sussmenn (2) has produced a number of stable, aggregateless mutants that remain permanently in the vegetative stage as single ameboid cells.’ One of these mutants was used in the present study in order to avoid the complicating factors of morphological transition. Previous investigation of the lipids of Dictyostelium diecoideum and its aggregateless mutant Agg 204 had been limited to the characterisation of a steroid, AZ-stigmasten3&ol, and the demonstration of its biosynthesis from acetate and mevalonate (3). Stigmastenol is produced in large quantities by Dictyostelium disco&&urn and is felt to be related to the activity of the aggregation-inducing factor, acrasin (4). The fatty acids and phosphatides of the cellular slime mold are described in this paper. The fatty acid composition of each of the phosphatides has been determined. Evidence is presented for considerable molecular heterogeneity within several classes of phosphatides. The phosphatides contain an unusually high percentage of unsaturated fatty acids. Of major interest is the presence, in large amounts, of three new dienoic long chain fatty acids whose structures have been established as 5,9-hexadecadienoic acid, 5,9-octadecadienoic acid, and 5,1 l-octadecadienoic acid. Under appropriate conditions of growth, two other new fatty acids, 9-heptadecenoic acid and 5,9-heptadecadienoic acid, normally present in small amounts, can be made to accumulate. Studies of the pathways of biosynthesis of these fatty acids are presented in an accompanying paper (5).

Synergistic morphogenesis by mixtures of Dictyostelium discoideum wild-type and aggregateless mutants.

SUMMARY: Aggregations involving mixtures of wild-type and ‘aggregateless’ mutant organisms of Dictyostelium discoideum were examined. The proportions of wild-type individuals which could initiate the formation of aggregative centres by their neighbours was found to depend markedly upon the particular mutant in the mixture. The mutants appear to have supplied some of the cells responding to the initiative stimulus where these were needed and also to have produced diffusible material enabling many more wild-type organisms to act as initiators than would ordinarily do so. The results also indicate the existence of a variety of cellular interactions which may underlie the normal morphogenetic sequence.