Streamer F mutants and chemotaxis of Dictyostelium.

Streamer F mutants have been found to be useful tools for studying the pathway of signal transduction leading to chemotactic cell movement. The primary defect in these mutants is in the structural gene for the cyclic GMP specific phosphodiesterase. This defect allows a larger and prolonged peak of cyclic GMP to be formed in response to the chemotactic stimulus, cyclic AMP. This characteristic aberrant pattern of cyclic GMP accumulation in the streamer F mutants has been correlated with similar patterns of changes in the influx of calcium from the medium, myosin II association with the cytoskeleton, myosin phosphorylation and a decrease in speed of movement of the amoebae. From these studies a sequence of events can be deduced that leads from cell surface cyclic AMP stimulation to cell polarization prior to movement of the amoebae in response to the chemotactic stimulus.     

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.     

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.     

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.     

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     

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.     

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.     

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.     

Cyclic AMP and calcium exchange in a cellular slime mold

Cyclic AMP is known to be an effective chemotactic agent for amebae of the cellular slime mold D. discoideum. A large amount of information from experiments on metazoa suggested that one cellular effect of cyclic AMP might be to alter the permeability of the cell membrane to Calcium or Sodium ions. On the basis of this information experiments were designed to test the effect of cyclic AMP on outflow of labeled Calcium or Sodium ions from amebae of D. discoideum. It was found that addition of cyclic AMP at 10^?4M resulted in a large increase of Ca⁴⁵ outflow from cells at the pre-aggregative or aggregative stage of development. No effect was found on Na²² outflow. It is suggested that this effect on Calcium permeability of the membrane is related to the chemotactic influence of ATP by some action on the contractile mechanism for ameboid movement. The phenomenon may be distinct from the enzyme inductive activity of cyclic AMP known for bacteria, and perhaps occurring in the cellular slime molds as well.     

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.     

Histamine modulation of eosinophil migration.

Preincubation of eosinophils with 10(-5) M or higher concentrations of histamine inhibited the eosinophil chemotactic response to endotoxin-activated serum whether by using the nucleopore filter assay and counting the cells migrating through the filter, or by using the Zigmond-Hirsch assay and counting the cells at each 10-mum interval. When the H2-receptor sites on the eosinophils were blocked by metiamide, the inhibitory capacity of histamine was prevented. Preincubation of eosinophils with 10(-6) M histamine increased the number of responding eosinophils to endotoxin-activated serum and this enhancement was blocked by an H1-receptor antagonist. Isoproteronol and aminophylline inhibited eosinophil movement and increasing concentrations of dibutryl cyclic AMP inhibited eosinophil migration. Concentrations of histamine that consistently resulted in inhibition of eosinophil movement stimulated an increase in cyclic AMP that was prevented by blocking the H2-receptor but not the H1-receptor. Thus, histamine-dependent inhibition of the eosinophil chemotactic response to other agents is mediated through the H2-receptor and is associated with an increase in the intracellular level of cyclic AMP whereas histamine dependent enhancement of eosinophil migration to other agents appears to be mediated through the H1-receptor. Eosinophils behave as a heterogeneous population as assessed by the ability of histamine to augment or inhibit cell migration. This may reflect differences in H1 to H2 receptor density or cell responsiveness to receptor stimulation. The chemoattractant activity of histamine itself is not influenced by H1 or H2 receptor antagonists, thus it is possible that an eosinophil has a third type of histamine receptor.     

Transmethylation inhibitors decrease chemotactic sensitivity and delay cell aggregation in Dictyostelium discoideum

In Dictyostelium discoideum, extracellular cyclic AMP (cAMP) induces chemotaxis and cell aggregation. Suspensions of cAMP-sensitive cells respond to a cAMP pulse with a rapid, transient increase of protein carboxyl methylation. The transmethylation inhibitors cycloleucine, L-homocysteine thiolactone, and coformycin decrease chemotactic sensitivity and delay cell aggregation when administered in concentrations which do not influence cAMP binding to cell surface receptors or the activity of total phosphodiesterase. The ability of the drugs to inhibit chemotaxis could be correlated with their capacity to convert the initial transient positive response of carboxyl methylation to cAMP into a negative one. This suggests that both protein O-methyltransferase and protein methylesterase are activated after stimulation of aggregative cells with cAMP, the net effect being a transient, positive response of methylation. In the presence of a sufficiently large dose of inhibitor, methyltransferase is inhibited, whereas methylesterase activity is much less affected, so that a transient negative response of methylation to cAMP is observed. The slow, positive response of carboxyl methylation to cAMP which occurs ca. 2.5 to 5 min after stimulus administration is not affected by inhibitors of transmethylation. These results suggest that methylation reactions are involved in the chemotactic response of D. discoideum cells to cAMP.     

Morphogenetic cell movement in Dictyostelium

Dictyostelium morphogenesis starts with the chemotactic aggregation of starving individual cells. The cells move in response to propagating waves of the chemoattractant cyclic AMP initiated by cells in the aggregation centre. During aggregation the cells begin to differentiate into several types with different signalling and chemotactic properties. These cell types sort out from each other to form an axial pattern in the slug. There is now good evidence that periodic chemotactic signals not only control aggregation, but also later stages of morphogenesis. These signals take the form of target patterns, spirals, multi-armed spirals and scroll waves. I will discuss their role in the control of cell movement during mound and slug formation and in the formation of the fruiting body.     

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.     

Suppression of cyclic AMP-induced cell excitation in Dictyostelium discoideum by inhibitors of eicosanoid oxidation

Abstract Receptor-mediated stimulation of Dictyostelium cells by the aggregative chemoattractant cyclic AMP leads to a complex excitatory response resulting in chemotaxis and the synthesis and release of cyclic AMP as the relayed chemotactic signal. However, the mechanism of this stimulus-response coupling is not well understood. In this study, we show that a number of compounds, best known as inhibitors of cyclooxygenase activity in mammalian cells, prevent cyclic AMP receptor-mediated cell excitation and cyclic AMP accumulation in aggregation-competent Dictyostelium cells. These observations suggest that some eicosanoid-like compound(s) may be involved in stimulus-response coupling in this organism, as is the case in higher eukaryotic cells.     

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.     

A model for cell type localization in the migrating slug of <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis> based on differential chemotactic sensitivity to cAMP and differential sensitivity to suppression of chemotaxis by ammonia

The three basic cell types in the migrating slug of Dictyostelium discoideum show differential chemotactic response to cyclic AMP (cAMP) and differential sensitivity to suppression of the chemotaxis by ammonia. The values of these parameters indicate a progressive maturation of chemotactic properties during the transdifferentiation of slug cell types. We present a model that explains the localization of the three cell types within the slug based on these chemotactic differences and on the maturation of their chemotactic properties.     

Reciprocal periodicity in cyclic AMP binding and phosphorylation of differentiating Dictyostelium discoideum cells.

The binding of [³H]cyclic AMP to cell surface receptors of differentiated D. discoideum cells at 25° is an oscillatory process with a periodicity of 2 min. This alternating change in the cells' binding capacity for cyclic AMP may be the basis for the refractory period to cyclic AMP stimulation, an essential feature of the chemotactic system. The incorporation of [³²P] by whole cells from [γ³²P]ATP is also oscillatory with a periodicity identical to that of [³H]cyclic AMP binding. However, the two processes are inversely related in time such that periods of maximal cyclic AMP binding correspond to periods of minimal cellular phosphorylation. These results suggest a receptor kinase/phosphatase mediated desensitization of the cyclic AMP receptor.     

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.