Calcium,cyclic GMP and the control of myosin II during chemotactic signal transduction ofDictyostelium

Evidence is presented for Ca²⁺ and cyclic GMP being involved in signal transduction between the cell surface cyclic AMP receptors and cytoskeletal myosin II involved in chemotactic cell movement. Ca²⁺ is shown to be required for chemotactic aggregation of amoebae. The evidence for uptake and/or eflux of this ion being regulated by the nucleotide cyclic GMP is discussed. The connection between Ca²⁺, cyclic GMP and chemotactic cell movement has been explored using “streamer F” mutants. The primary defect in these mutants is in the structural gene for the cyclic GMP-specific phosphodiesterase which results in the mutants producing an abnormally prolonged peak of accumulation of cyclic GMP in response to stimulation with the chernoattractant cyclic AMP. While events associated with production and relay of cyclic AMP signals are normal, certain events associated with movement are (like the cyclic GMP response) abnormally prolonged in the mutants. These events include Ca²⁺ uptake, myosin II association with the cytoskeleton and inhibition of myosin heavy and light chain phosphorylation. These changes can be correlated with the amoebae becoming elongated and transiently decreasing their locomotive speed after chemotactic stimulation. Other mutants studied in which the accumulation of cyclic GMP in response to cyclic AMP stimulation was absent produced no myosin II responses. Models are described in which cyclic GMP (directly or indirectly via Ca²⁺) regulates accumulation of myosin II on the cytoskeleton by inhibiting phosphorylation of the myosin heavy and light chain kinases.     

Signal transduction and motility ofDictyostelium

This review is concerned with the roles of cyclic GMP and Ca²⁺ ions in signal transduction for chemotaxis ofDictyostelium. These molecules are involved in signalling between the cell surface cyclic AMP receptors and cytoskeletal myosin II involved in chemotactic cell movement. Evidence is presented for uptake and/or eflux of Ca²⁺ being regulated by cyclic GMP. The link between Ca²⁺, cyclic GMP and chemotactic cell movement has been explored using streamer F mutants whose primary defect is in the structural gene for the cyclic GMP-specific phosphodiesterase. This mutation causes the mutants to produce an abnormally prolonged peak of cyclic GMP accumulation in response to stimulation with the chemoattractant cyclic AMP. The production and relay of cyclic AMP signals is normal in these mutants, but certain events associated with movement are (like the cyclic GMP response) abnormally prolonged in the mutants. These events include Ca²⁺ uptake, myosin II association with the cytoskeleton and regulation of both myosin heavy and light chain phosphorylation. These changes can be correlated with changes in the shape of the amoebae after chemotactic stimulation. Other mutants in which the accumulation of cyclic GMP in response to cyclic AMP stimulation was absent produced no myosin II responses.A model is described in which cyclic GMP (directly or indirectly via Ca²⁺) regulates accumulation of myosin II on the cytoskeleton by regulating phosphorylation of the myosin heavy and light chain kinases.     

Signal transduction for chemotaxis in Dictyostelium amoebae

The signal for chemotaxis in D. discoideum is cyclic AMP. This molecule binds to cell surface receptors and triggers the production of inositol (1,4,5)trisphosphate which releases Ca2+ from non-mitochondrial stores. The subsequent chain of signal transduction events brings about the polymerization of cytoskeletal actin (associated with pseudopodium formation) within five seconds and the formation of a peak of cyclic GMP within 10 s. Evidence from streamer F mutants indicates that the cyclic GMP regulates the association of myosin with the cytoskeleton that occurs at 25-50 s and that this phenomenon is concerned with elongation of the amoebae during chemotactic movement.     

Streamers: chemotactic mutants of Dictyostelium discoideum with altered cyclic GMP metabolism

Mutants of Dictyostelium discoideum that developed huge aggregation streams in expanding clones were investigated using optical and biochemical techniques. Representatives of the six complementation groups previously identified (stmA-stmF) were found to be similar to the parental wild-type strain XP55 in both the extent and timing of their ability to initiate and relay chemotactic signals and in the formation of cyclic AMP receptors and phosphodiesterases. The mutants differed from the wild-type in producing an abnormal chemotactic (movement) response visible using both dark-field optics with synchronously aggregating amoebae on solid substrata and light scattering techniques with oxygenated cell suspensions. Mutants of complementation group stmF showed chemotactic movement responses lasting up to 520 s, rather than 100 s as seen in the parental and other strains. Measurements of cyclic GMP formed intracellularly in response to chemotactic pulses of cyclic AMP in stmF mutants showed that abnormally high concentrations of this nucleotide were formed within 10 s and were not rapidly degraded. A causal correlation between defective cyclic GMP metabolism and the altered chemotactic response is suggested, and a model is proposed that accounts for the formation of huge aggregation streams in clones of these mutants.U     

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.     

Inositol 1,4,5-trisphosphate induces cyclic GMP formation in Dictyostelium discoideum

Chemotactic signalling in the cellular slime mould Dictyostelium discoideum employs signalling molecules such as folate and cyclic AMP. These bind to specific cell surface receptors and rapidly trigger internal responses that induce chemotactic movement of the amoebae. Previous studies have shown that actin is polymerised within 3-5 sec of cyclic AMP or folate binding and that a peak of cyclic GMP is formed within 9-12 sec. Release of Ca2+ from intracellular stores has been implicated as a secondary messenger. Here we present evidence that D-myo-inositol 1,4,5-trisphosphate, when added to permeabilized amoebae of Dictyostelium, can mimic the action of chemoattractants on normal intact amoebae in inducing cyclic GMP formation. Our data suggest that IP3, which is known to act as an intermediary messenger between cell surface hormone receptors and release of Ca2+ from internal stores in mammalian cells, functions in a similar capacity during chemotaxis of this primitive eukaryote.     

Calcium induces cyclic GMP formation in Dictyostelium

Cyclic GMP is rapidly formed a few seconds after binding of chemotactic signalling molecules to specific receptors on the cell surface of Dictyostelium amoebae. This phenomenon could be mimicked by addition of a pulse of Ca2+ to permeabilised amoebae. The concentration of Ca2+ for half-maximal response was 60 microM. Other ions (K+, Na+, Mg+ or Mn+) had no effect. A pulse of 5 microM IP3 produced a cyclic GMP response of similar magnitude but IP2 elicited no response. The data provide strong support for the hypothesis that cell surface receptor binding induces cyclic GMP formation by liberating Ca2+ from internal stores.     

Evidence for a Second Chemotactic System in the Cellular Slime Mold, Dictyostelium discoideum

An unknown substance found in bacteria (Escherichia coli) is especially effective in attracting the vegetative amoebae of the cellular slime mold, Dictyostelium discoideum. However, the aggregating amoebae are not attracted to it at all. On the other hand, the vegetative amoebae show very little chemotactic response to cyclic adenosine monophosphate (cyclic AMP), whereas the aggregating amoebae are exceptionally responsive to it. It is suggested that the new factor may be used in food seeking, whereas cyclic AMP, the chemotactic substance responsible for aggregation, is the acrasin of this species. The important point is that the amoebae are differentially stage-specific in their responses to these two chemotactic agents.     

Chemotactic response of wild-type and aggregation-defective mutants of Polysphondylium violaceum

Abstract. The aggregation-specific chemoattractant for Polysphondylium violaceum is N-propionyl-γ-L-glutamyl-L-ornithine-δ-lactam ethyl ester, or glorin. Wild-type amoebae allowed to develop in liquid culture acquire increased ability to respond to glorin shortly after starvation, i.e., just prior to the time they become aggregation competent. Similarly, as development proceeds, the amoebae show decreased sensitivity to folic acid, but they show almost no response to cyclic AMP at any time during their development in liquid culture. The optimum concentrations for the chemotactic response are 10^-8 M for glorin and 10^-5–10^-6 M for folic acid. A class of aggregation-defective mutants, aggA , will not aggregate in the absence of an excreted pheromone, D factor. During development in liquid culture in the presence or absence of D factor, these aggA mutants show a chemotactic response similar to that of wild-type amoebae to folic acid and glorin. However, D factor does enhance the chemotactic response of aggA mutants to glorin. In the absence of D factor, mutant amoebae will form fruiting bodies if exposed to a chemotactic gradient of either folic acid or glorin. Under these conditions, the mutant amoebae circumvent the requirement for D factor in order to develop.     

Adenylate cyclase is required for chemotaxis to phosphotransferase system sugars by Escherichia coli.

We report that in Escherichia coli, chemotaxis to sugars transported by the phosphotransferase system is mediated by adenylate cyclase, the nucleotide cyclase linked to the phosphotransferase system. We conclude that adenylate cyclase is required in this chemotaxis pathway because mutations in the cyclase gene (cya) eliminate or impair the response to phosphotransferase system sugars, even though other components of the phosphotransferase system known to be required for the detection of these sugars are relatively unaffected by such mutations. Moreover, merely supplying the mutant bacteria with the products of this enzyme, cyclic AMP and cyclic GMP, does not restore the chemotactic response. Because a residual chemotactic response is observed in certain strains with residual cyclic GMP synthesis but no cyclic AMP synthesis, it appears that the guanylate cyclase activity rather than the adenylate cyclase activity of the enzyme may be required for chemotaxis to sugars transported by the phosphotransferase system. Mutations in the cyclic nucleotide phosphodiesterase gene, which increase the level of both cyclic AMP and cyclic GMP, also reduce chemotaxis to these sugars. Therefore, it appears that control of the level of a cyclic nucleotide is critical for the chemotactic response to phosphotransferase system sugars.     

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.     

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 min 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 lenght 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°C, aggregation begins later, chemotaxis takes effect over a greater distance, and the maximal binding activity is higher than at 22°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.     

Evidence against direct involvement of cyclic GMP or cyclic AMP in bacterial chemotactic signaling.

Defects in phosphotransferase chemotaxis in cya and cpd mutants previously cited as evidence of a cyclic GMP or cyclic AMP intermediate in signal transduction were not reproduced in a study of chemotaxis in Escherichia coli and Salmonella typhimurium. In cya mutants, which lack adenylate cyclase, the addition of cyclic AMP was required for synthesis of proteins that were necessary for phosphotransferase transport and chemotaxis. However, the induced cells retained normal phosphotransferase chemotaxis after cyclic AMP was removed. Phosphotransferase chemotaxis was normal in a cpd mutant of S. typhimurium that has elevated levels of cyclic GMP and cyclic AMP. S. typhimurium crr mutants are deficient in enzyme III glucose, which is a component of the glucose transport system, and a regulator of adenylate cyclase. After preincubation with cyclic AMP, the crr mutants were deficient in enzyme II glucose-mediated transport and chemotaxis, but other chemotactic responses were normal. It is concluded that cyclic GMP does not determine the frequency of tumbling and is probably not a component of the transduction pathway. The only known role of cyclic AMP is in the synthesis of some proteins that are subject to catabolite repression.     

In vitro stimulation of neutrophil motility by levamisole: maintenance of cgmp levels in chemotactically stimulated levamisole-treated neutrophils.

Levamisole at concentrations of 10(-3) M or 10(-4) M consistently increased neutrophil random motility and chemokinesis (stimulated random migration). Similar concentrations also increased directional movement of polymorphonuclear leukocytes to both endotoxin-activated serum and hydrolyzed casein. This effect on chemotaxis was due to a true stimulation and was not due solely to increased random movement. The effect of levamisole on the neutrophils could be removed by washing, but persisted if the cells were initially treated with levamisole and serum or endotoxin-activated serum. After neutrophil stimulation with chemotactic factor an initial rise in intracellular cyclic AMP levels was detected which was not influenced by prior levamisole treatment. Intracellular cyclic GMP levels after an initial slight depression, returned to resting levels and gradually diminished over a 60-minute period. Levamisole-treated cells consistently showed higher cyclic GMP levels and it is postulated that by maintaining intracellular cyclic GMP levels, microtubular assembly and cell motility might be enhanced.     

Guanidine hydrochloride-induced shedding of a Dictyostelium discoideum plasma membrane fraction enriched in the cyclic adenosine 3',5'-monophosphate receptor

The cell surface cyclic AMP receptor of Dictyostelium discoideum is under study in a number of laboratories with respect to both its role in development of the organism and the physiology of excitation-response coupling. We report here that when starved amoebae are exposed to the chaotrope guanidine hydrochloride at 1.8 M, they shed a particulate cyclic AMP binding activity into the medium. This activity is due to membrane vesicles which originate from the cell surface. The vesicles are enriched up to 150-fold in cyclic AMP binding activity and up to 14-fold in phospholipid content when compared to the starting amoebae. The cyclic AMP binding activity of the membrane vesicles is identical to that of the cell surface receptor with respect to the following properties; (i) it is lacking in preparations from unstarved, vegetative amoebae; (ii) it is not inhibited by cyclic GMP and is stimulated by calcium ions; (iii) it has very rapid rates of association and dissociation of bound cyclic AMP; (iv) it has two classes of binding sites with dissociation constants similar to those of the surface receptors of whole amoebae. The binding activity of the isolated membranes is stable for several days at 4 degrees C and the lower affinity binding sites are stable up to several months when stored at -80 degrees C. Due to enrichment and stability of the receptor in this preparation, it should be highly suitable for many types of studies. The usefulness is enhanced by the fact that the preparation does not contain detectable cyclic AMP phosphodiesterase activity.     

A possible involvement of cya gene in the synthesis of cyclic guanosine 3':5'-monophosphate in E. coli.

Based on the following genetical experiments, the cya gene in E. coli was shown to be involved in the synthesis of both cyclic AMP and cyclic GMP. First, all five independent cya-deficient mutants accumulated exceedingly low amounts of cyclic GMP. Second, the ability to form both cyclic AMP and cyclic GMP was simultaneously restored by transduction of an intact cya locus to one of the above cya-deficient mutants. Third, a spontaneous revertant from one of the above mutants regained the synthetic activity for cyclic GMP as well as for cyclic AMP. Fourth, the characteristic of a strain overproducing cyclic GMP was co-transduced with the cya locus. These results suggest that the synthesis of both cyclic GMP and cyclic AMP is mediated by the same enzyme, adenylate cyclase, Interestingly, a reciprocal effect of glucose starvation was observed on the accumulation of both cyclic nucleotides. The formation of cyclic AMP was greatly enhanced on glucose starvation, whereas that of cyclic GMP proceeded at a slower rate than in the presence of glucose. This effect was observed only in cells carrying normal cya and crp genes, but not in a cya-altered or a crp-deficient strain.     

The role of cyclic AMP in the chemotactic responsiveness and spontaneous motility of rabbit peritoneal neutrophils. The inhibition of neutrophil movement and the elevation of cyclic AMP levels by catecholamines, prostaglandins, theophylline and cholera toxin.

Agents known to affect intracellular levels of cyclic AMP in many diverse systems have been tested for their effect on the chemotaxis induced by Escherichia coli culture filtrates, spontaneous motility and cyclic AMP levels of rabbit peritoneal neutrophils. Prostaglandin E1 and A1 but not prostaglandin F2alpha increased neutrophil cyclic AMP levels and, correspondingly, only the former two prostaglandins inhibited chemotaxis. Nevertheless, a quantitative relationship between prostaglandin stimulation of cyclic AMP and inhibition of chemotaxis could not be found. Epinephrine, isoproterenol, and, to a much lesser extent, norepinephrine increased neutrophil cyclic AMP through beta adrenergic stimulation. Only epinephrine and isoproterenol inhibited chemotaxis, but the inhibition was variable and not related to the ability of these catecholamines to increase intracellular cyclic AMP. Cholera toxin increased neutrophil cyclic AMP after a 30-min lag period which paralled its inhibitory effect on chemotaxis and spontaneous motility. However, the effect on chemotaxis require 50 ng/ml of toxin whereas the effect on cyclic AMP was manifested at 2 ng/ml of toxin. Prior to 30-min preincubation there was no effect of even 1250 ng/ml of toxin on either cyclic AMP or chemotaxis. Choleragenoid prevented the effects of toxin on both cyclic AMP and chemotaxis. The bacterial chemotactic factor obtained from E. coli culture filtrates did not effect a measurable change in levels of neutrophil cyclic AMP. The data indicate that even though cyclic AMP is not, in the main sequence of events, triggering the chemotactic response, increases in neutrophil cyclic AMP may modulate the movement and thus the chemotactic responsiveness of the neutrophil.     

Elevated cyclic GMP concentrations in rabbit bone marrow culture and mouse spleen following erythropoietic stimulation.

The effects of erythropoietin and hypoxia on cyclic nucleotide concentrations in erythroid tissue were evaluated. A rabbit bone marrow culture system and a mouse spleen model provided evidence that erythropoietin and an hypoxic stimulus which increases erythropoietin production may enhance erythropoiesis by initiating reciprocal changes in erythroid cell cyclic nucleotide levels. Cyclic GMP appears to be the active signal in mediating the response to erythropoietin, whereas cyclic AMP may be a passive signal allowing full expression of the cyclic GMP response. Whether the type of response mediated by cyclic nucleotides is proliferative, differentiative or both is not clear, but our data and that of other investigators suggest that cyclic GMP mediates the proliferative actions of erythropoietin.     

On a mathematical model describing the aggregation of amoebae

In this paper an extension of a mathematical model of Keller and Segel (1970) describing the aggregation of amoebae is presented. In their paper (Keller and Segel, 1970) they showed that the onset of the aggregation could be viewed as a spatial instability. Their instability condition involved diffusion constants of the cyclic AMP and of the amoebae as well as a constant describing the chemotactic behavior of the amoebae. In our case we consider a temporal instability that depends only on the kinetics of cyclic AMP production, degradation and transport through the cell wall. Our model then explains the oscillatory behavior of the cyclic AMP in well-stirred suspensions of amoebae. In addition we discuss existence and non-existence of nonuniform steady states of the nonlinear parabolic system involved.     

ROLE OF CYCLIC AMP IN THE POLARIZED MOVEMENT OF THE MIGRATING PSEUDOPLASMODIUM OF DICTYOSTELIUM DISCOIDEUM

Cyclic AMP is known to act as a chemotactic agent that directs the movement of aggregating Dictyostelium discoideum cells. Its role in the multicellular organization of this organism was studied with special reference to the polarized movement of the migrating pseudoplasmodium (slug). The results showed that the tip of the slug has the ability to function as an aggregation center, and that slug cells are chemotactically sensitive to cyclic AMP. The addition of calcium or magnesium appeared to enhance formation of cell streams, thus facilitating detection of chemotactic response of slug cells, but this addition was not required for the response itself. These indicate that the polar movement of the slug may be principally controlled by cyclic AMP.