Mutants of thermotaxis in Dictyostelium discoideum

Amoebae of Dictyostelium discoideum, strain HL50 were mutagenized with N-methyl-N′-nitro-N-nitrosoguanidine, cloned, allowed to form pseudoplasmodia and screened for aberrant positive and negative thermotaxis. Three types of mutants were found. Mutant HO428 exhibits only positive thermotaxis over the entire temperature range (no negative thermotaxis). HO596 and HO813 exhibit weakened positive thermotaxis and normal negative thermotaxis. The weakened positive thermotactic response results in a shift toward warmer temperatures in the transition temperature from negative to positive thermotaxis. Mutant HO209 exhibits weakened positive and negative thermotactic responses and has a transition temperature similar to the ‘wild type’ (HL50). The two types of mutants represented by HO428, HO596 and HO813 support the model that positive and negative thermotaxis have separate pathways for temperature sensing. The type of mutants which contains HO209 suggests that those two pathways converge at some point before the response.     

Correlations between prestalk-prespore tendencies and cAMP-related activities in Dictyostelium discoideum

Amoebae of the cellular slime mould Dictyostelium discoideum (strain Ax2) grown in axenic medium containing 86 mM glucose [G(+) cells] or no glucose [G(−) cells] were examined for the characteristics of aggregation, cAMP secretion rate, cAMP phosphodiesterase activity and cell surface cAMP-binding activity. (When G(+) and G(−) cells are mixed, G(+) cells preferentially differentiate into prespore cells and sort out to the posterior region of a slug.)Under the same conditions, G(−) cells aggregate later than G(+) cells and the formation of stable streams by G(−) cells was particularly delayed. The movements of G(−) cells during aggregation were less organized compared with the ordered behaviour of G(+) cells, yet G(−) cells seemed to be more sensitive to chemotactic signals. Furthermore, the cAMP-binding activity of G(−) cells was considerably higher than that of G(+) cells, a difference that is probably due to the difference in the number of the cAMP receptor sites. Also G(−) cells, compared to G(+) cells, secreted slightly more cAMP and showed higher activity of cell-bound phosphodiesterase activity at the aggregation stage, whereas the extracellular phosphodiesterase activity was lower, although these differences were minor.     

Gene regulation during dedifferentiation in Dictyostelium discoideum

During development of Dictyostelium discoideum, cells acquire the capacity to rapidly recapitulate morphogenesis. Therefore, when cells at the loose aggregate stage are disaggregated and challenged to reaggregate, they do so in a tenth of the original time. If loose aggregate cells are disaggregated and resuspended in buffered dextrose solution (erasure medium), they retain the capacity of rapid recapitulation for 80 min, then completely lose this capacity in a single, synchronous step referred to as the "erasure event." The erasure event sets in motion a program of dedifferentiation during which cells lose developmentally acquired characteristics at different times. The erasure event is inhibited by the addition of 10(-4) M cAMP to erasure medium. The synthesis of 33 growth-associated polypeptides, the synthesis of 53 development-associated polypeptides, and the level of 2 development-associated RNAs have been monitored during the erasure program and in cultures inhibited from erasing by the addition of 10(-4) M cAMP. Growth-associated polypeptides begin to be resynthesized and development-associated polypeptides exhibit dramatic decreases in rate of synthesis at different times throughout the first 240 min in erasure medium. Inhibiting the erasure event with cAMP has no major effect in the resynthesis of the majority of growth-associated polypeptides. Only one growth-associated polypeptide, V28, is completely inhibited by cAMP, suggesting that it may play a role in the erasure process. In contrast, inhibiting the erasure event with cAMP has a marked effect on the synthesis of development-associated polypeptides, causing a dramatic reduction in the rate at which synthesis decreases for 6 polypeptides, and completely inhibits the decrease in the synthetic rate of 8 polypeptides. The two development-associated RNAs, 16G1 and 10C3, exhibit two distinctly different patterns of loss during erasure, but in both cases cAMP added at time zero of the erasure process dramatically retards or inhibits loss. In addition, when cAMP is added just prior to the erasure event, it inhibits the erasure event and stimulates a rapid increase in the level of 16G1 RNA back to the developmental level. The level of 16G1 RNA then remains at this level for at least 400 min. When cAMP is added after the erasure event, it causes a low, transient increase in the level of 16G1 RNA. These results are considered both in relation to the program of erasure, and in relation to the role of cAMP in the expression of developmental genes during the forward program of development.     

A phototaxis signalling complex in Dictyostelium discoideum

Phototaxis has been studied in a variety of organisms belonging to all three major taxonomic domains – the bacteria, the archaea and the eukarya. Dictyostelium discoideum is one of a small number of eukaryotic organisms which are amenable to studying the signalling pathways involved in phototaxis. In this study we provide evidence based on protein coimmunoprecipitation for a phototaxis signalling complex in Dictyostelium that includes the proteins RasD, filamin, ErkB, GRP125 and PKB.     

Genes involved in Dictyostelium discoideum sexual reproduction

Macrocyst formation in the cellular slime moulds is a sexual process induced under dark and humid conditions. Normal development life cycle in these organisms involves proliferation by cell division and, upon starvation, formation of multicellular aggregates and fruiting bodies, consisting of spores and stalk cells. Macrocyst formation, cell division by binary fission and spore formation are thus three alternative modes of reproduction, for which it is of interest to understand how a choice is made. The genetic basis of asexual development and fruiting body formation is well known, by contrast information on the genetic control of sexual reproduction during macrocyst formation is scarce. In Dictyostelium discoideum, the most widely used species, several cell-surface proteins relevant to sexual cell fusion have been identified using cell fusion-blocking antibodies, but isolation of the relevant genes has been unsuccessful. Analysis of sexually deficient mutants, some of which are normal for asexual development, has shown that sexual reproduction is regulated by both specific genes and genes that are also involved in asexual development. Reverse genetic analysis of 24 genes highly enriched in a gamete-specific subtraction library has revealed four genes involved in the regulation of sexual cell interactions. One of them was found to be a novel regulator of the cAMP signalling pathway specific to sexual development. Studies on the molecular genetic control of the sexual cycle will be reviewed and their contribution to our understanding of the organization and function of the D. discoideum genome as a whole discussed.     

Isolation of mutations in Dictyostelium discoideum affecting α-mannosidase

We have isolated mutant strains of the cellular slime mold, Dictyostelium discoideum, which specifically lack α-mannosidase-1. This enzyme accumulates during the developmental phase of the life cycle. A minor isozyme, α-mannosidase-2, was recognized which accumulates only during culmination and makes up about 10 p. cent of the total activity in fruiting bodies. The minor isozyme has a pH response distinctly different from the major isozyme. Loss of the major isozyme, α-mannosidase-1, does not disrupt the normal sequence of biochemical differentiations.     

Cytochrome b of the Dictyostelium discoideum plasma membrane

Cytochrome b of the plasma membrane of Dictyostelium discoideum was investigated in purified plasma membranes and in solubilized form. The membrane-bound cytochrome b can be reduced by NADH. This reduction is inhibited by p-hydroxymercuribenzoate. The reduced cytochrome b does not react with carbon monoxide. Its apparent molecular weight lies between 13000 and 16000. Tryptic digestion yields a large, heme-containing peptide with an apparent molecular weight between 12000 and 15000. After solubilization with cholate, cytochrome b can be enriched by reversed-phase HPLC, indicating that it contains also a hydrophobic component. With these properties, cytochrome b of the D. discoideum plasma membrane resembles microsomal cytochrome b₅.     

A stage-specific inhibitor of adenylate cyclase in Dictyostelium discoideum

A sudden increase in adenylate cyclase activity occurs during the chemotaxis and aggregation of Dictyostelium discoideum. Preincubation of extracts from the pre-aggregation stage, in which adenylate cyclase activity was low, with post-aggregation stages, in which the increase in activity occurred, resulted in the demonstration of a heat-stable inhibitor of adenylate cyclase (ACI) that was present only during the early stages of development. Cellular fractionation studies showed that ACI was present in both the 100 000 g pellet and supernatant fractions. The inhibitor was not inactivated by proteases or protease inhibitors. A heat-treated preparation of the inhibitor was dialysable. The effect of ACI was dependent upon a pre-incubation treatment, with notable inhibition occurring only after a 20 min pre-incubation period. The apparent inhibition was not artifactual, due to the degradation of the substrate, ATP, or to the loss of the reaction product, cAMP. Additionally, the inhibitor was specific for adenylate cyclase, as it had no effect on the activity of several other enzymes, including cAMP phosphodiesterase.     

Utilization of trehalose during Dictyostelium discoideum spore germination

An analysis of metabolism by measurement of respiratory quotient values indicates that reduced substances, such as lipids and/or amino acids, are the primary respiratory substrates of dormant Dictyostelium discoideum spores. The spores appear to consume both reduced substances and carbohydrates during the swelling stage of germination. The respiration of emerged myxamoebae is again dominated by the consumption of reduced substances. The pool of trehalose remains largely intact during heat-induced activation and also during postactivation lag. The initiation of spore swelling is accompanied by a decrease in the trehalose pool; the majority of trehalose is consumed before late spore swelling. Upon placing heat-activated spores under restrictive environmental conditions, swelling and trehalose hydrolysis are both prevented. Release from these conditions results in rapid swelling and hydrolysis of trehalose. Trehalase, the enzyme responsible for trehalose breakdown, is present in dormant spores at basal levels. This preformed enzyme is responsible for the hydrolysis of trehalose even though there is a significant increase in trehalase activity with the emergence of myxamoebae. RNA and protein synthesis inhibitors do not prevent trehalose hydrolysis or spore swelling. It is concluded that oxidation of reduced substances occurs in dormant, activated, and swollen spores, as well as in emerged myxamoebae of D. discoideum. Carbohydrate utilization dominates over the oxidation of reduced substances only during the swelling stage of germination.     

Dictyostelium discoideum chemotaxis: Threshold for directed motion

The chemotactic response of Dictyostelium discoideum cells to stationary, linear gradients of cyclic adenosine 3′,5′-monophosphate (cAMP) was studied using microfluidic devices. In shallow gradients of less than 10⁻³ nM/μm, the cells showed no directional response and exhibited a constant basal motility. In steeper gradients, cells moved up the gradient on average. The chemotactic speed and the motility increased with increasing steepness up to a plateau at around 10⁻¹ nM/μm. In very steep gradients, above 10 nM/μm, the cells lost directionality and the motility returned to the sub-threshold level. In the regime of optimal response the difference in receptor occupancy at the front and back of the cell is estimated to be only about 100 molecules.     

An autonomously propagating luciferase-encoding vector for Dictyostelium discoideum

We have constructed a luc reporter vector for Dictyostelium discoideum using a 626-bp fragment from the nuclear-associated plasmid Ddp2. The ori from Ddp2 is localized within this fragment and was used to provide an autonomous replication sequence for the reporter vector. This reporter vector was stably retained in D. discoideum AX3K cells without alteration. The vector molecule was also found to exist in relatively low copy number compared to other Dictyostelium vectors in the transformed cells. We demonstrated the utility of this vector as a reporter vector with glycogen synthase promoter/luc fusions of varying sizes.     

In situ localization of actin mRNA in Dictyostelium discoideum aggregates

By in situ hybridization of a labeled cloned DNA to cellular RNA, we have studied the distribution of actin mRNA in differentiating Dictyostelium discoideum aggregates. In migrating pseudoplasmodia, the mRNA is distributed uniformly. However, in culminating aggregates (sorogens) actin mRNA is enriched in stalk cells and in the cells at the anterior tip. Thus, the preferential synthesis of actin in prestalk and stalk cells, previously reported, is due at least partially to increased levels of actin mRNA.     

Characterization of a glycosyl-phosphatidylinositol degrading activity in Dictyostelium discoideum membranes

Antigen 117 is a glycolipid-anchored cell surface protein implicated in cell-cell cohesion of Dictyostelium discoideum amoebae. Previous studies have demonstrated that during cell aggregation some of the protein is released from the cell surface. Here we report the characterization of the enzymatic activity involved in the 117 antigen release. The data indicate that the releasing enzyme is a phosphatidylinositol phospholipase C. The data also indicate that structural features of glycolipid anchors are conserved in a variety of organisms.     

The endocytosis of concanavalin A by Dictyostelium discoideum cells

Differentiating cells of D. discoideum in suspension bind ConA. The proportion of the bound lectin which is competitively removed by methyl-α- mannopyranoside decreases with the time of exposure. Ferritin conjugated ConA is seen to bind both to the cell surface and to be taken into the cells, the proportion of the ConA inside the cells increasing with time. The surface bound ConA is removed by washing with methyl-α- mannopyranoside while the endocytosed ConA appears unaffected. It is suggested that much of the [¹²⁵I]ConA, uncompetable by methyl-α- mannopyranoside in our and other binding studies, may be this intracellular ConA.     

An auto-inhibitor of zygote giant cell formation in Dictyostelium discoideum

The first observable event of sexual development in mated cultures (NC4x V12) of Dictyostelium discoideum is the appearance of zygote giant cells which are the product of cell fusions. The increase in the number of giant cells, which begins at about 21 h, is complete by 28 h. In this paper we show that a low molecular weight auto-inhibitor of zygote giant cell formation is present in 28-h cultures. The data presented suggest that this auto-inhibitor is a natural regulator of sexual development in D. discoideum.     

Analysis of ribosomal RNA metabolism during development of Dictyostelium discoideum

Using double labelling protocols we have compared the developmental metabolism of ribosomal subunits fabricated during vegetative growth of Dictyostelium discoideum with those accumulated during subsequent development. Unlike vegetative growth when ribosomal subunits are accumulated in equal amounts, early development is characterized by the accumulation of approximately twice as much large as small subunit. The unusual paucity of small subunit was not due to selective sequestration by the nucleus as previously thought nor cytoplasmic degradation. Ribosomal subunits, whether synthesized during growth or development, were degraded at equivalent rates by the developing cell indicating the lack of preferential conservation at the degradative level.     

The activation of cell aggregation by phosphorylation in Dictyostelium discoideum

Single amoebae of D. discoideum are phosphorylated in the presence of external ATP. Phosphorylation is catalyzed by a cAMP independent cell membrane bound protein kinase. As a result of phosphorylation cell aggregation is induced and the chemotactic sensitivity of the amoebae to a cAMP gradient decreased. Cell membrane phosphorylation may be involved in triggering cell aggregation in vivo. The fact that the number of free phosphorylable sites per cell decreases at the onset of aggregation gives support to this hypothesis. The existence of a plasma membrane bound phosphoprotein phosphatase suggests a possible regulator role for this enzyme on the phosphorylation of the amoebae. Finally, ATP inhibits intercellular contact sites outside the aggregation center. Despite this inhibiting effect on cell adhesiveness, amoebal movement toward an aggregation center maintains its normal periodicity.     

Construction of an extrachromosomally replicating transformation vector for Dictyostelium discoideum

An extrachromosomally replicating transformation vector for Dictyostelium discoideum has been constructed using sequences of the endogenous Dictyostelium plasmid Ddp2. This transformation vector pnDeI (9.6 kb) replicates as a high copy number plasmid in Dictyostelium and is located in the nucleus. It has been constructed as shuttle vector containing the Escherichia coli vector pUC19 for replication and selection in E. coli and a part of the Tn903 transposon which confers resistance to G418 for selection in Dictyostelium. In order to show that the vector can be used for cloning and stable propagation of Dictyostelium DNA, a fragment of the Dictyostelium alpha-actinin gene that was marked with a synthetic oligonucleotide was cloned into pnDeI and found to be stably maintained in the extrachromosomal vector without undergoing noticeable recombination with the endogenous gene.