José M. Mato

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).

Guanylate cyclase activation in response to chemotactic stimulation in Dictyostelium discoideum

In aggregating cells ofDictyostelium discoideum cyclic AMP induces chemotaxis [1 ] and the secretion of cyclic AMP after a brief activation of adenylate cyclase [2]. Cyclic AMP binds to cell surface receptors [3] and subsequently causes a fast transient increase in the cellular cyclic GMP content which accompanies pseudopod formation [4,5] and precedes cyclic AMP synthesis [5]. This increase in the level of cyclic GMP can occur as a result of an increased synthesis and/or inhibited degradation of the cyclic nucleotide. We show here that cyclic AMP induces a rapid 3--6-fold transient stimulation of guanylate cyclase in aggregation competent cells of D. discoideum. Also we found that physiological concentrations of ATPenhance the basal enzyme activity.

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.

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]

CHEMOTACTIC SIGNAL AND CYCLIC GMP ACCUMULATION IN DICTYOSTELIUM

Publisher Summary This chapter focuses on chemotactic signal and cyclic GMP accumulation in Dictyostelium. The chemotactic signals in Dictyostelium discoideum are released periodically from aggregation centers and propagated from cell to cell. Cyclic adenosine monophosphate (cAMP) mediates cell aggregation by acting as chemotactic attractant and as propagator of the signal. The signal for chemotaxis is a spatial gradient of cAMP concentration measured by cell surface receptors. The translation of the chemotactic signal into pseudopod formation toward the attractant source is mediated by changes in the levels of a regulatory molecule that probably is cGMP. The chapter also discusses the specificity of the cAMP chemoreceptor.

CHEMOTAXIS IN DICTYOSTELIUM DISCOIDEUM: EFFECT OF CONCANAVALIN A ON CHEMOATTRACTANT MEDIATED CYCLIC GMP ACCUMULATION AND LIGHT SCATTERING DECREASE

In cells of the cellular slime mold Dictyostelium discoideum concanavalin A (Con A), at a concentration of 100 microgram per ml, inhibits folic acid and cyclic AMP induced decrease in light scattering. Con A has no effect on folic acid mediated cyclic GMP accumulation and increases cyclic AMP mediated cyclic GMP accumulation two-fold. At a lower Con A concentration, 10 microgram per ml, changes in light scattering induced by folic acid are normal and cyclic AMP induces a monophasic instead of a biphasic response. The stimulatory effect of Con A on cyclic AMP mediated cyclic GMP accumulation is still observable at 10 microgram Con A per ml. When cells are repeatedly stimulated with cyclic AMP, a decrease in light scattering without being accompanied by changes in cyclic GMP concentration is observed. Based on these results a model for chemotaxis is proposed.

An acrasin-like attractant from yeast extract specific for Dictyostelium lacteum.

The transition of the unicellular to the multicellular stage in Dictyostelium lacteum is not mediated by cyclic AMP. The attractant for aggregative amoebae of this cellular slime mold species was isolated from yeast extract and purified more than 1000-fold without a significant loss of activity. Several characteristics of the chemotactic molecule specific for D. lacteum are reported, and the presence of an inactivating enzyme has been demonstrated.

The chemotactic activity of cyclic AMP and AMP derivatives with substitutions in the phosphate moiety in Dictyostelium discoideum

Cyclic AMP mediates cell aggregation in Dictyostelium disco&urn [ 1 ] . During aggregationcompetence differentiation, amoebae develop a cellsurface-bound cyclic AMP chemoreceptor [2-51. The interaction of, cyclic AMP with the chemoreceptor can be studied by triggering cells in their sensitive stage with an external source of cyclic AMP and monitoring the amoebae response [6]. By means of this technique it has been concluded that the cyclic AMP chemoreceptor in D. discoideum is very sensitive to structural changes in the cyclic AMP molecule specially at the phosphate moiety [7] , Also, it has been suggested that at the aggregative stage of this species only cyclic nucieotides show chemotactic acitivity [8]. In this paper further evidence is given on the essential role of the phosphate moiety in the interaction of cyclic AMP with the chemoreceptor. Surprisingly chemotactic activity is found with several AMP derivatives despite their non-cyclic structure. These results are discussed in the light of our present knowledge on chemotaxis in D. discoideum.

S-Adenosylmethionine inhibits Ca2+ uptake into Escherichia coli membrane vesicles

We have recently observed that the addition of Sadenosylmethionine (SAM) inhibits ATP-dependent 45Ca2’ uptake into Dictyostelium discoideum vesicles (J. M. M., M. Cao, in preparation). Whereas nothing is known about the function of SAM in Dictyostelium, there is a well established relationship between protein methylation and the processing of chemotactic stimuli in bacteria [ 11. Further, inverted membrane vesicles isolated from bacteria are known to accumulate 45Ca2+ in the presence of ATP [2,3]. Therefore, we decided to examine the effect of SAM on 45CaZI uptake into Escherichia coli vesicles. We show that 1 mM SAM inhibits by -7-fold the uptake of 45CaZ+ into E. coli vesicles prepared by lysis with a French pressure cell.

ATP increases chemoattractant induced cyclic GMP accumulation in Dictyostelium discoideum

Abstract Changes in guanosine cyclic 3′,5′-monophosphate associated with adenosine cyclic 3′,5′-monophosphate and folic acid addition in the presence of ATP have been examined in Dictyostelium discoideum . Preincubation with 1 mM ATP had no effect on the basal cyclic GMP level but increased the cycli GMP accumulation in response to cylci AMP (5·10 −8 M) or folic acid (5·10 −6 M) 40–50%. ATP could not be replaced by ADP of 5′-adenylyliminodiphosphate. Because ATP has no effect on cyclic AMP receptor binding these results indicate that structural membrane alterations (e.g. membrane phosphorylation) may control the transduction of a chemotactic signal.