Biochemical differentiation in a mutant of Dictyostelium discoideum defective in cyclic AMP chemotaxis and in intercellular cohesion

A temperature-sensitive mutant of Dictyostelium discoideum has been isolated based on its lack of chemotaxis toward cyclic AMP at the restrictive temperature, 27 degrees C. The mutant develops normally at the permissive temperature, 22 degrees C, but fails to aggregate or complete development at the restrictive temperature. The temperature-sensitive phenotype can be bypassed by allowing cultures to grown into late log phase or to starve for 60-90 min at 22 degrees C prior to a shift to 27 degrees C. At 27 degrees C, the mutant overproduces cell surface cyclic AMP receptors of both high and low affinity and is capable of spontaneous oscillations in light scattering in cell suspensions. Despite its complete lack of morphological development, the mutant undergoes extensive biochemical differentiation. At the onset of starvation, it shows increased levels of N-acetylglucosaminidase, it express cyclic AMP receptors at the normal time and, although somewhat slowly, suppresses those receptors as if aggregation had been achieved. Metabolic pulse labellings with [35S]methionine revealed that the mutant at 27 degrees C displays the same changes in the patterns of newly synthesized proteins observed during the vegetative-to-aggregation and the aggregation-to-slug stages of normal development. The only clear difference from wild type was the failure of the culmination-stage isozyme of beta-glucosidase to appear. The mutant is defective in establishment of intercellular cohesion mechanisms, correlated with poor agglutination by concanavalin A, at the restrictive temperature. The properties of the mutant place severe constraints on models regarding the role of chemoreception and intercellular cohesion in regulation of gene expression.     

Selection of chemotaxis mutants of Dictyostelium discoideum

A method has been developed for the efficient selection of chemotaxis mutants of Dictyostelium discoideum. Mutants defective in the chemotactic response to folate could be enriched up to 30-fold in one round of selection using a chamber in which a compartment that contained the chemoattractant was separated by a sandwich of four nitrocellulose filters from a compartment that contained buffer. Mutagenized cells were placed in the center of the filter layer and exposed to the attractant gradient built up between the compartments for a period of 3-4 h. While wild-type cells moved through the filters in a wave towards the compartment that contained attractant, mutant cells remained in the filter to which they were applied. After several repetitions of the selection procedure, mutants defective in chemotaxis made up 10% of the total cell population retained in that filter. Mutants exhibiting three types of alterations were collected: motility mutants with either reduced speed of movement, or altered rates of turning; a single mutant defective in production of the attractant-degrading enzyme, folate deaminase; and mutants with normal motility but reduced chemotactic responsiveness. One mutant showed drastically reduced sensitivity in folate-induced cGMP production. Morphogenetic alterations of mutants defective in folate chemotaxis are described.     

EppA, a putative substrate of DdERK2, regulates cyclic AMP relay and chemotaxis in Dictyostelium discoideum

The mitogen-activated protein kinase DdERK2 is critical for cyclic AMP (cAMP) relay and chemotaxis to cAMP and folate, but the details downstream of DdERK2 are unclear. To search for targets of DdERK2 in Dictyostelium discoideum, 32PO4(3-)-labeled protein samples from wild-type and Dderk2- cells were resolved by 2-dimensional electrophoresis. Mass spectrometry was used to identify a novel 45-kDa protein, named EppA (ERK2-dependent phosphoprotein A), as a substrate of DdERK2 in Dictyostelium. Mutation of potential DdERK2 phosphorylation sites demonstrated that phosphorylation on serine 250 of EppA is DdERK2 dependent. Changing serine 250 to alanine delayed development of Dictyostelium and reduced Dictyostelium chemotaxis to cAMP. Although overexpression of EppA had no significant effect on the development or chemotaxis of Dictyostelium, disruption of the eppA gene led to delayed development and reduced chemotactic responses to both cAMP and folate. Both eppA gene disruption and overexpression of EppA carrying the serine 250-to-alanine mutation led to inhibition of intracellular cAMP accumulation in response to chemoattractant cAMP, a pivotal process in Dictyostelium chemotaxis and development. Our studies indicate that EppA regulates extracellular cAMP-induced signal relay and chemotaxis of Dictyostelium.     

Cyclic AMP oscillations in suspensions of Dictyostelium discoideum

A model developed previously for signal relay and adaptation in the cellular slime mould Dictyostelium discoideum is shown to account for the observed oscillations of calcium and cyclic AMP in cellular suspensions. A qualitative argument is given which explains how the oscillations arise, and numerical computations show how characteristics such as the period and amplitude of the periodic solutions depend on parameters in the model. Several extensions of the basic model are investigated, including the effect of cell aggregation and the effect of time delays in the activation and adaptation processes. The dynamics of mixed cell populations in which only a small fraction of the cells are capable of autonomous oscillation are also studied.     

Four key signaling pathways mediating chemotaxis in Dictyostelium discoideum

Chemotaxis is the ability of cells to move in the direction of an external gradient of signaling molecules. Cells are guided by actin-filled protrusions in the front, whereas myosin filaments retract the rear of the cell. Previous work demonstrated that chemotaxis of unpolarized amoeboid Dictyostelium discoideum cells is mediated by two parallel pathways, phosphoinositide-3-kinase (PI3K) and phospholipase A2 (PLA2). Here, we show that polarized cells exhibit very good chemotaxis with inhibited PI3K and PLA2 activity. Using genetic screens, we demonstrate that this activity is mediated by a soluble guanylyl cyclase, providing two signals. The protein localizes to the leading edge where it interacts with actin filaments, whereas the cyclic guanosine monophosphate product induces myosin filaments in the rear of the cell. We conclude that chemotaxis is mediated by multiple signaling pathways regulating protrusions at the front and rear of the cell. Cells that express only rear activity are polarized but do not exhibit chemotaxis, whereas cells with only front signaling are unpolarized but undergo chemotaxis.     

Extracellular calmodulin regulates growth and cAMP-mediated chemotaxis in Dictyostelium discoideum

Zymocin and PaT are killer toxins that induce cell cycle arrest of sensitive yeast cells in G1 and S phase, respectively. Recent studies have revealed that these two toxins cleave specific tRNAs, indicating that the cell growth impairment is due to the tRNA cleavage. Additionally, we have previously shown that the active domain of colicin D (D-CRD), which also cleaves specific Escherichia coli tRNAs, statically impairs growth when expressed in yeast cells. To verify that phase-specific cell cycle arrest is also induced by the expression of D-CRD, D-CRD and the subunits of zymocin and PaT that have tRNA cleaving activity were expressed in yeast cells and cell cycle status was analyzed. Our results indicate that phase-specific arrest does not commonly occur by tRNA cleavage, and it saves the cell viability. Furthermore, the extent of protein synthesis impairment may determine the phase specificity of cell cycle arrest.     

Local and spatially coordinated movements in Dictyostelium discoideum amoebae during chemotaxis

We have studied chemotaxis by individual Dictyostelium discoideum amoebae using strong, local gradients of the chemoattractant cyclic AMP. Gradients were provided by diffusion of cyclic AMP from a microneedle, which could be positioned at various points around the cell. Responses to changes in the gradient indicate how the cell is structurally organized for chemotactic movement. There is a polarity in the responsiveness of the surface to stimulation by cyclic AMP along the length of the amoeba. Furthermore, two aspects of chemotactic movement can be distinguished. The first response to cyclic AMP is a locally generated extension of a hyaline pseudopod from the region of the surface nearest the stimulus. The second response, the flow of cytoplasm in the direction of the stimulus, is coordinated and separate from the first response. The coordination appears to depend on the nucleus or on the microtubule-organizing center.     

Cellular responsiveness to cyclic AMP in a phosphodiesterase-defective mutant of Dictyostelium discoideum

Responsiveness of Dictyostelium discoideum amoebae to cAMP, a chemotactic mediator, was investigated in a strain defective in cAMP-phosphodiesterase production. Cells were subjected to a high cAMP signal (10(-6) M) in the presence or absence of exogenous phosphodiesterase, and the changes of intracellular cAMP and cGMP concentrations and of adenylate cyclase activity were measured. In the presence of cAMP hydrolysis, both adenylate and guanylate cyclases are transiently activated. In the absence of hydrolysis, the high and constant extracellular cAMP concentration is sufficient to elicit a re-activation of adenylate cyclase a few minutes after the first transient response. In contrast, levels of cGMP remain basal for at least 20 min after termination of the initial response to the cAMP addition.     

Pseudopodium activation and inhibition signals in chemotaxis by Dictyostelium discoideum amoebae

The behaviour of Dictyostelium discoideum amoebae has been studied in natural cAMP waves and in controlled spatial and temporal gradients. Chemoattractant gradients induce responses which indicate that amoebae spatially compare concentration increases at different points on the cell surface. This allows them to respond to the relative spatial and temporal gradients in a manner that is little affected by the absolute attractant concentration over several orders of magnitude. The changes in turning behaviour, motility and morphology that are induced by attractant gradients are consistent with transduction of stimuli into two intracellular signals - one activating and the other inhibiting pseudopodium formation. The former measures the present attractant concentration at particular points on the cell surface - the local, current signal. The latter measures the average attractant concentration over the whole cell surface during the recent past - the global, past signal. Both signals may be part of a normal pseudopodium autoactivation and inhibition system responsible for amoeboid morphology and motility. Attractants could modulate this system to generate the complex behavioural responses observed.     

Evidence for chemotaxis during sexual development in Dictyostelium discoideum.

Amoebae in mated cultures of Dictyostelium discoideum show oriented movement towards young aggregates, suggesting that cemotaxis is involved in macrocyst development. Amoebae also show directional movement towards midendocyte stages, indicating that as the macrocyst develops it continues to be a source of chemoattractant. These data are discussed in terms of our current knowledge about mating in the cellular slime moulds.     

Constitutively active protein kinase A disrupts motility and chemotaxis in Dictyostelium discoideum

The deletion of the gene for the regulatory subunit of protein kinase A (PKA) results in constitutively active PKA in the pkaR mutant. To investigate the role of PKA in the basic motile behavior and chemotaxis of Dictyostelium discoideum, pkaR mutant cells were subjected to computer-assisted two- and three-dimensional motion analysis. pkaR mutant cells crawled at only half the speed of wild-type cells in buffer, chemotaxed in spatial gradients of cyclic AMP (cAMP) but with reduced efficiency, were incapable of suppressing lateral pseudopods in the front of temporal waves of cAMP, a requirement for natural chemotaxis, did not exhibit the normal velocity surge in response to the front of a wave, and were incapable of chemotaxing toward an aggregation center in natural waves generated by wild-type cells that made up the majority of cells in mixed cultures. Many of the behavioral defects appeared to be the result of the constitutively ovoid shape of the pkaR mutant cells, which forced the dominant pseudopod off the substratum and to the top of the cell body. The behavioral abnormalities that pkaR mutant cells shared with regA mutant cells are discussed by considering the pathway ERK2 —| RegA —| [cAMP] → PKA, which emanates from the front of a wave. The results demonstrate that cells must suppress PKA activity in order to elongate along a substratum, suppress lateral-pseudopod formation, and crawl and chemotax efficiently. The results also implicate PKA activation in dismantling cell polarity at the peak and in the back of a natural cAMP wave.     

Localization and levels of cyclic AMP during development of Dictyostelium discoideum

Cyclic AMP functions as the chemotactic signal during aggregation of amoebae of the cellular slime mold Dictyostelium discoideum. Evidence suggests that cAMP also acts as a regulatory molecule during Dictyostelium multicellular differentiation. We have used ultramicrotechniques and a sensitive radioimmunoassay to measure the levels of cAMP within the culmination stage individual. We show that there is a peak of cAMP at the culmination stage of development and that in the individual at this stage the molecule is localized in a gradient within the spore mass.     

Quantitative analysis of cyclic AMP waves mediating aggregation in Dictyostelium discoideum

We have previously reported the detection of cAMP waves within monolayers of aggregating Dictyostelium discoideum cells (K. J. Tomchik and P.N. Devreotes, 1981, Science 212, 443-446). The computer-assisted analysis presented here of the fluorographic images of the cAMP waves reveals (1) all the waves have a consistent width and height; (2) cAMP concentrations within centers of concentric aggregation territories oscillate periodically while at spiral centers the concentration builds up to a plateau value within 2 mm; (3) cells within the region of intersection of two oppositely directed cAMP waves are stimulated to produce more cAMP than those responding to a single wave; (4) cells start to move when the cAMP level begins to increase and cease movement when the peak cAMP concentration reaches the cell.     

Purification and characterization of developmentally regulated AMP deaminase from Dictyostelium discoideum

AMP deaminase, the enzyme that catalyzes the conversion of adenosine monophosphate (AMP) to inosine monophosphate (IMP) and ammonia, was purified from the cellular slime mold, Dictyostelium discoideum in the nutrient-deprived state. The native enzyme had an apparent molecular weight of 199,000 daltons. Its apparent Km was 1.6 mM and its Vmax was 1.0 mumol min-1 mg-1, as measured by the release of IMP From AMP. The enzyme, like other AMP deaminases, was found to be activated by ATP, and inhibited either by GTP or inorganic phosphate. It was also specific for the deamination of AMP. Deaminase activity was increased either when vegetative cells were placed in a nutrient-deprived medium (for up to 6 h) or when vegetative cells were treated with the drug hadacidin. In cells actively growing in complete media, enzyme activity was more non-specific, hydrolyzing adenosine as well as AMP. AMP deaminase in D. discoideum appears to be stage-specific and developmentally regulated, possibly serving to regulate the adenylated nucleotide pool and the interconversion to guanylated nucleotides during early morphodifferentiation.     

Purification and characterization of the extracellular cyclic AMP phosphodiesterase of Dictyostelium discoideum

Extracellular phosphodiesterase for adenosine 3':5'-monophosphate [EC 3.1.4.17] was purified from the supernatant of aggregation phase culture of Dictyostelium discoideum, and two types (type I and type II) of the enzyme were found. The type I enzyme was not absorbed on DEAE-Sephacel at pH 8.5 and had an apparent molecular weight of about 67,000 daltons. In contrast, the type II enzyme was adsorbed on DEAE-Sephacel and had an apparent molecular weight of about 120,000 daltons. The Km values of the two types were similar (2-4 microM). Upon SDS polyacrylamide gel electrophoresis analyses, however, both types produced the same bands with molecular weights of 55,000 and 57,000, indicating that they are two different forms composed of common constituents. During the growth phase, the two types of the enzyme were present in culture supernatant in roughly equal amounts, but type II accumulated predominantly in the aggregation phase, suggesting that the ratio of activity of the two forms is under developmental control. Rabbit antiserum prepared against purified type II enzyme cross-reacted with type I as well as membrane-bound enzyme, indicating that the three classes of the enzyme possess some common sequence.