Theo M. Konijn

Cyclic AMP and folic acid mediated cyclic GMP accumulation in Dictyostelium discoideum

In a suitable environment spores of the Dictyostelia [ 1 ] germinate yielding small amoebae. Each amoebae feeds on bacteria and divides. Following consumption of the food supply amoebae aggregate forming a slug where cells differentiate into spores and stalk ceils. The orientation of cells towards a food source or an aggregation center is guided by gradients of concentration of chemotactic molecules. In D. discoideum vegetative amoebae are attracted by folic acid [2] and aggregative amoebae by cAMP [3]. The sensory reception of a cAMP signal involves binding of cAMP to cell-surface bound receptors [4-7] and analysis of the signal in terms of changes of concentration over distance [8]. The sensory system transmits the signal to pseudopod formation into the direction of the attractant within t0 s [9]. Recently we have shown that, at physiological concentrations, cAMP induces an increase in the level of cGMP that precedes pseudopod formation [10]. cGMP peaks are high (up to 10-fold with 5 X 10 -7 M cAMP) and brief (pre-stimulation level is reached in about 25-30 s after addition of cAMP).

Signal Transduction in the Cellular Slime Molds

Intercellular communication in higher organisms depends on the central nervous system and hormones. Simple organisms such as the cellular slime molds communicate intercellularly only by using hormone-like signals. The most intensively studied species of the cellular slime molds is Dictyostelium discoideum. Aggregating cells of this species secrete cyclic AMP as chemoattractant, and very low concentrations of this intercellular communication signal induce molecular, behavioral and developmental changes in neighboring cells. The transduction of such a signal in the responding cell has several characteristics in common with hormone action. Binding of cyclic AMP to the cell-surface receptors of the responding cell is specific, rapid, saturable and reversible. The activated receptor regulates internal cGMP and CAMP levels and, as after hormone activation, calcium fluxes, methylation, refractory periods and down regulation are observed. Moreover, the synthesis of key enzymes might be a response to chemotactic signals. Dictyostelium, as well as being simple, is also a suitable organism to grow in large quantities and, experimentally, it is a favorable subject because the amebae are activated by chemoattractants in their single-cell phase. A large variety of mutants that may be blocked somewhere between the beginning and the end of the transduction process is available. When the cells are still free, uptake of food takes place by phagocytizing bacteria. Also, the vegetative cells respond to simple chemoattractants, such as folic acid (Pan et al., 1972), that are secreted by the bacteria. Specificity for chemotactic signals is not so important in this stage because the amebae feed on almost any bacterial species (Raper, 1937). After the food supply is exhausted and there is no gradient of chemoattractant secreted by bacteria, the cells themselves start to secrete more specific chemotactic

Chemotaxis and binding of cyclic AMP in cellular slime molds.

To obtain more information about how cyclic AMP mediates cell aggregation as found in some species of the cellular slime molds, we determined the maximal binding activity of cyclic AMP in different species under various environmental conditions. The binding of cyclic AMP is limited to amoebae using this cyclic nucleotide as chemotactic agent. Maximal binding activity proved to coincide with a maximal chemotactic response and to be related to the length of the period between the vegetative and the aggregative phase. Of the species studied, Dictyostelium discoideum has the highest cellular density of cyclic AMP receptors and is the most sensitive to cyclic AMP as attractant. At 15 degrees C, aggregation begins later, chemotaxis takes effect over a greater distance, and the maximal binding activity is higher than 22 degrees C. The number of cyclic AMP receptors is independent of temperature. The delay in the onset of aggregation and the increased chemotactic response in darkness is not due to a change in the maximal binding activity. The binding of cyclic AMP and its inactivation is discussed in the light of cell aggregation.

Identification of the acrasin of Dictyostelium minutum as a derivative of folic acid.

The acrasin of the slime mold Dictyostelium minutum was isolated from aggregating cells and purified. The compound was species specific and more active in the aggregative than in the vegetative stage. Three observations strongly suggest a structural relationship between the acrasin and folic acid. (1) Folic acid inhibited acrasin degradation by D. minutum. (2) Methotrexate, an antagonist of chemotaxis towards folic acid, also inhibited the response to the acrasin. (3) The chemotactic response to an excess of folic acid was delayed. The response was also delayed to simultaneously tested low amounts of a related compound, but not to unrelated compounds (Van Haastert, 1982). The response to the acrasin was observed to be delayed by excess of folic acid. The acrasinase was identified as a folic acid C9-N10 splitting enzyme. Based on chromatographic properties and biological activity of the acrasin and folate derivatives, the chemical structure of the acrasin is discussed.

Antagonists of chemoattractants reveal separate receptors for cAMP, folic acid and pterin in Dictyostelium

Abstract Adenosine 3′,5′-monophosphate (cAMP), folic acid and pterin are chemoattractants in the cellular slime molds. The cAMP analog, 3′-amino-cAMP, inhibits a chemotactic reaction to cAMP at a concentration at which the analog is chemotactically inactive. The antagonistic effect of 3′-amino-cAMP on the chemotactic activity of cAMP is competitive, which suggests that 3′-amino-cAMP antagonizes cAMP via the chemotactic receptor for cAMP. 3′-Amino-cAMP does not antagonize folic acid or pterin. The binding of folic acid to post-vegetative Dictyostelium discoideum cells is inhibited by low concentrations of 2-deamino-2-hydro folic acid (DAFA [7]). DAFA is neither chemotactically active, nor does it inhibit a chemotactic reaction to folic acid. This questions the involvement of the main folic acid cell surface-binding sites in the chemotactic response to folic acid. The pterin analog, 6-aminopterin, is an antagonist of pterin, but not of cAMP or folic acid. Our results show that cAMP, folic acid and pterin are detected by different receptors. Furthermore, they suggest that the antagonistic action of 3′-amino-cAMP and 6-aminopterin is localized in the signal transduction pathway at a step before the signals from the separate receptors have arrived at a single pathway.

Unified control of chemotaxis and cAMP mediated cGMP accumulation by cAMP in Dictyostelium discoideum.

Summary Guanosine 3′5′ cyclic monophosphate (cGMP) accumulation in response to adenosine 3′5′ cyclic monophosphate (cAMP) in the aggregateless Dictyostelium discoideum mutant, Agip 55, has been investigated in view of cGMPs proposed role during chemotaxis. This mutant was deficient in chemotaxis to cAMP and also showed a deficient cGMP accumulation in response to this attractant. A deficient chemotactic response can be reverted in Agip 55 cells into a normal chemotactic response by pulsating with cAMP. cAMP pulsated cells showed a higher cAMP binding capacity and also gave a normal cGMP accumulation in response to cAMP. Therefore it is concluded that the same cAMP receptor controls chemotaxis and cGMP accumulation. These results show also that cAMP controls the development of cAMP receptors and cAMP mediated cGMP accumulation and that the chemotactic response involves changes in the cGMP levels.

Chemotaxis in the cellular slime molds: I. The effect of temperature

The effect of temperature on chemotaxis in the cellular slime mold Dictyostelium discoideum has been studied by incubating small populations of washed myxamoebae at different temperatures. Droplets containing a cell suspension of known density were deposited on a hydrophobic agar surface. The myxamoebae normally stayed within the boundaries of the implanted droplets, but they moved outside the margins of such droplets when they were attracted by acrasin secreted by neighboring populations. Sensitive cells in the responding populations were mainly attracted between the beginning of aggregation and its completion in the attracting populations. Secretion of acrasin before the formation of compact centers of aggregation was demonstrated at low temperatures and with time lapse photography. A decrease in temperature was correlated with: (1) an increase in the distance of attraction; (2) an increase in the time interval between the beginning of aggregation and its completion; and (3) an increase in the lapse of time between the deposition of the myxamoebae and the beginning of aggregation. The chronological age of the attracting cells did not seem to affect the chemotactic response. The increased attraction at lower temperatures was correlated with a longer period between early aggregation and its completion. Only the temperature during aggregation had an effect on the distance over which cells could be oriented; any temperature change applied during the preaggregative stages was of no consequence to the attraction at later stages.

Relationships between the ligand specificity of cell surface folate binding sites, folate degrading enzymes and cellular responses in Dictyostelium discoideum

The affinity of four distinct types of folate binding sites and of two membrane-bound folate degrading enzymes for 15 folic acid derivatives was monitored. Apart from two types (AH and AL of binding sites, all binding classes or enzymes show different affinity patterns. This strongly suggests the observed binding sites to be nonidentical to the membrane-bound folate deaminase or folate C9-N10 cleaving enzyme. The analog specificity of the chemotactic response towards folates shows a strong resemblance to the specificity of binding to one of the binding classes: ‘B-sites’ (p < 0.01%). Less or no correlation was observed between the other classes or the enzymes and the chemotactic response. This may indicate that the B-sites are involved in the transduction of an extracellular signal to chemotactic cell movement. Folates elicit secretion of cAMP. Recently, the activity of several folate derivatives to evoke a cAMP response was studied (Devreotes, P.N. (1983) Dev. Biol. 95, 154–162). Comparison of this activity and the specificity of the binding sites in this study, suggests that the ‘A-sites’ are involved in the cAMP response.

Cyclic GMP Binding Activity in Dictyostelium discoideum

In addition to these short term effects, CAMP also has long term effects controlling the pro- gram for cell differentiation. Thus, the application of CAMP in pulses shorten the time interval between starvation and cell aggregation by speeding up the appearance of cell to cell contact sites A [6]

Chapter 16 Measurement of Chemotaxis in Dictyostelium

Publisher Summary The molecular mechanism of chemotaxis contributes to the explanation of signal transduction. To investigate the transfer of chemotactic signals one has to measure the response to chemoattractants. A shift from observing cell behavior to exploring the mechanism of signal transduction required sophisticated chemotactic assays. Measurement of simple qualitative positive or negative responses is supplemented with semiquantitative observation of chemotaxis. Several assays are developed; some have already been applied to other cells, and others are specifically designed to measure a chemotactic response in the slime molds. The first chemotaxis assays for cellular slime molds are meant to prove that amoebae move chemotactically to the center of an aggregate. These qualitative assays are gradually succeeded by semiquantitative assays when chemoattractants are isolated, purified, characterized, and sometimes identified. The further development of assays is directed to the elucidation of cell behavior in gradients of chemoattractants. A considerable extension of automation of data acquisition is presented. This automation allows new assays to be performed which focus both on the cell population and on individual cells in chemotactic gradients with defined temporal and spatial components.