Gradient Of Cyclic AMP Research Articles

Possible roles of the endocytic cycle in cell motility

Starving, highly motile Dictyostelium cells maintain an active endocytic cycle, taking up their surface about every 11 minutes. Cell motility depends on a functional NSF (N-ethylmaleimide sensitive factor) protein--also essential for endocytosis and membrane trafficking generally--and we, therefore, investigated possible ways in which the endocytic cycle might be required for cell movement. First, NSF, and presumably membrane trafficking, are not required for the initial polarization of the leading edge in a cyclic-AMP gradient. Second, we can detect no evidence for membrane flow from the leading edge, as photobleached or photoactivated marks in the plasma membrane move forward roughly in step with the leading edge, rather than backwards from it. Third, we find that the surface area of a cell--measured from confocal reconstructions--constantly fluctuates during movement as it projects pseudopodia and otherwise changes shape; increases of 20-30% can often occur over a few minutes. These fluctuations cannot be explained by reciprocal changes in filopodial surface area and they substantially exceed the 2-3% by which membranes can stretch. We propose that the endocytic cycle has a key function in motility by allowing adjustment of cell surface area to match changes in shape and that, without this function, movement is severely impaired.

Blebbing of Dictyostelium cells in response to chemoattractant

Stimulation of Dictyostelium cells with a high uniform concentration of the chemoattractant cyclic-AMP induces a series of morphological changes, including cell rounding and subsequent extension of pseudopodia in random directions. Here we report that cyclic-AMP also elicits blebs and analyse their mechanism of formation. The surface area and volume of cells remain constant during blebbing indicating that blebs form by the redistribution of cytoplasm and plasma membrane rather than the exocytosis of internal membrane coupled to a swelling of the cell. Blebbing occurs immediately after a rapid rise and fall in submembraneous F-actin, but the blebs themselves contain little F-actin as they expand. A mutant with a partially inactivated Arp2/3 complex has a greatly reduced rise in F-actin content, yet shows a large increase in blebbing. This suggests that bleb formation is not enhanced by the preceding actin dynamics, but is actually inhibited by them. In contrast, cells that lack myosin-II completely fail to bleb. We conclude that bleb expansion is likely to be driven by hydrostatic pressure produced by cortical contraction involving myosin-II. As blebs are induced by chemoattractant, we speculate that hydrostatic pressure is one of the forces driving pseudopod extension during movement up a gradient of cyclic-AMP.

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 perpendicular RegA perpendicular [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.

Chemotaxis to cAMP and slug migration in Dictyostelium both depend on migA, a BTB protein.

Chemotaxis in natural aggregation territories and in a chamber with an imposed gradient of cyclic AMP (cAMP) was found to be defective in a mutant strain of Dictyostelium discoideum that forms slugs unable to migrate. This strain was selected from a population of cells mutagenized by random insertion of plasmids facilitated by introduction of restriction enzyme (a method termed restriction enzyme-mediated integration). We picked this strain because it formed small misshapen fruiting bodies. After isolation of portions of the gene as regions flanking the inserted plasmid, we were able to regenerate the original genetic defect in a fresh host and show that it is responsible for the developmental defects. Transformation of this recapitulated mutant strain with a construct carrying the full-length migA gene and its upstream regulatory region rescued the defects. The sequence of the full-length gene revealed that it encodes a novel protein with a BTB domain near the N terminus that may be involved in protein-protein interactions. The migA gene is expressed at low levels in all cells during aggregation and then appears to be restricted to prestalk cells as a consequence of rapid turnover in prespore cells. Although migA- cells have a dramatically reduced chemotactic index to cAMP and an abnormal pattern of aggregation in natural waves of cAMP, they are completely normal in size, shape, and ability to translocate in the absence of any chemotactic signal. They respond behaviorally to the rapid addition of high levels of cAMP in a manner indicative of intact circuitry connecting receptor occupancy to restructuring of the cytoskeleton. Actin polymerization in response to cAMP is also normal in the mutant cells. The defects at both the aggregation and slug stage are cell autonomous. The MigA protein therefore is necessary for efficiently assessing chemical gradients, and its absence results in defective chemotaxis and slug migration.

The regulation of chemotaxis and chemokinesis in Dictyostelium amoebae by temporal signals and spatial gradients of cyclic AMP

The tactic and kinetic locomotion of Dictyostelium discoideum amoebae were examined in cyclic AMP (cAMP) spatial gradient and temporal signal fields. The distributions of migrating cells were examined within 150 microns-thick micropore filters after incubation with different cAMP concentrations, [cAMP], applied in three ways across the fields: as positively or negatively developing gradients, generated either by increasing or decreasing the [cAMP] on one side of the filter, respectively, or as static, linear gradients after negative development. Chemotaxis was only induced by oriented, temporally increasing [cAMP]. Pulses propagated by molecular diffusion or mechanical flow were equally effective. Negatively developing cAMP gradients had no initial effect on cell accumulation. However, if the subsequent static spatial gradient was maintained by an infusion system, some gradients also induced cell accumulation, whose degree and direction depended on the gradient [cAMP]. The basis of this new effect was examined by tracking individual cells by computer-assisted videomicroscopy during locomotion in different [cAMP]. Cells produced a triphasic [cAMP]-dependent response, with optimal cell motility induced by 10-30 nM. The results demonstrate that cell accumulation either up-field or down-field in spatial gradients is governed by the field locations of the attractant concentrations that induce the relative locomotory maxima and minima in the gradient field. Cells perceive the ambient [cAMP], but cannot read the spatial gradient orientation in static or yet steeper regions of developing gradients. Accumulation in static spatial gradients is a function of klino- and orthokinesis, but chemotaxis requires an oriented cAMP pulse or impulse.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of presumptive morphogens on prestalk and prespore cell gene expression in monolayers of Dictyostelium discoideum

Abstract The expression of three prestalk cell-specific genes ( ecmA, ecmB and pDd26) was examined during in vitro differentiation in cell monolayers, in an attempt to explain the spatial heterogeneity of the prestalk region of migrating Dictyostelium pseudoplasmodia. Under these conditions ecmA, ecmB and pDd26 mRNAs were expressed sequentially in response to the addition of differentiation inducing factor-1 (DIF)-1, a temporal sequence similar to that observed during normal development. ecmA and ecmB mRNAs reached a maximum level 2–4 h after DIF-1 supplementation and then declined, whereas pDd26 mRNA levels increased more slowly but remained high 24 h after DIF addition. The increases in expression in response to increasing concentrations of either DIF-1 or DIF-2 were identical for the three genes, suggesting that neither alteration in DIF concentration nor species was an important determinant of spatial heterogeneity. Ammonia had the same inhibitory effect on the expression of all three prestalk cell-specific genes and stimulated the expression of the prespore cell-specific gene, D19. These results indicate that ammonia is also not responsible for the spatial heterogeneity of the prestalk cell region. In contrast, cyclic AMP had a differential effect on the expression of the prestalk cell specific genes: ecmA expression was variably stimulated, pDd26 expression was inhibited and ecmB expression was sometimes stimulated and sometimes inhibited. These results are difficult to explain in terms of a gradient of cyclic AMP in the prestalk region. We postulate that temporal responses are more important than spatial responses to cyclic AMP in regulating stalk cell differentiation.

Modulation of testicular galactolipid sulphotransferase activity by phosphorylation. Stimulation of enzyme activity in vitro by an endogenous kinase.

Evidence is presented for a testicular protein kinase activity capable of stimulating the activity in vitro of a partially purified preparation of the testicular galactolipid sulphotransferase. This enzyme is responsible for the synthesis of the major mammalian testicular glycolipid, sulphogalactosylglycerol, and is an early marker of differentiation during spermatogenesis. This stimulatory activity has been separated by affinity chromatography, using 3',5'-ADP-agarose, from both the detergent-solubilized microsomes (microsomal fractions) and the soluble fraction of the testicular homogenate. The stimulator was eluted from the affinity matrix by either a salt, or, more selectively, a cyclic AMP gradient. Thus this matrix can function as an analogue of 3',5'-cyclic AMP. The activity of the sulphotransferase stimulator was ATP-dependent and coincident with protein kinase activity. Sulphotransferase activity was also stimulated in the presence of commercial preparations of cyclic AMP-dependent protein kinase and stimulation was prevented in the presence of kinase inhibitors. Our results suggest that sulphogalactolipid biosynthesis is regulated by a phosphorylation process during spermatogenesis. In addition, our results suggest that affinity chromatography on 3',5'-ADP-agarose may provide a general method for the purification of cyclic AMP-dependent kinases.

A Dictyostelium mutant deficient in severin, an F-actin fragmenting protein, shows normal motility and chemotaxis.

A severin deficient mutant of Dictyostelium discoideum has been isolated by the use of colony immunoblotting after chemical mutagenesis. In homogenates of wild-type cells, severin is easily detected as a very active F-actin fragmenting protein. Tests for severin in the mutant, HG1132, included viscometry for the assay of F-actin fragmentation in fractions from DEAE-cellulose columns, labeling of blots with monoclonal and polyclonal antibodies, and immunofluorescent-labeling of cryosections. Severin could not be detected in the mutant using these methods. The mutation in HG1132 is recessive and has been mapped to linkage group VII. The mutant failed to produce the normal severin mRNA, but small amounts of a transcript that was approximately 100 bases larger than the wild-type mRNA were detected in the mutant throughout all stages of development. On the DNA level a new Mbo II restriction site was found in the mutant within the coding region of the severin gene. The severin deficient mutant cells grew at an approximately normal rate, aggregated and formed fruiting bodies with viable spores. By the use of an image processing system, speed of cell movement, turning rates, and precision of chemotactic orientation in a stable gradient of cyclic AMP were quantitated, and no significant differences between wild-type and mutant cells were found. Thus, under the culture conditions used, severin proved to be neither essential for growth of D. discoideum nor for any cell function that is important for aggregation or later development.

Chemoattraction and chemotaxis in Dictyostelium discoideum: myxamoeba cannot read spatial gradients of cyclic adenosine monophosphate.

Myxamoebae of the morphogenetic cellular slime mold Dictyostelium discoideum are thought to be able to accurately read and respond to directional information in spatial gradients of cyclic AMP. We examined the spatial and temporal mechanisms proposed for chemotaxis by comparing the behavior of spreading or evenly distributed cell populations after exposure to well-defined spatial gradients. The effects of gradient generation on cells were avoided by using predeveloped gradients. Qualitatively different responses were obtained using (a) isotropic, (b) static spatial, or (c) temporal (impulse) gradients in a simple chamber of penetrable micropore filters. We simulated models of chemotaxis and chemokinesis to aid our interpretations. The attractive and locomotory responses of populations were maximally stimulated by 0.05 microM cyclic AMP, provided that cellular phosphodiesterase was inhibited. But a single impulse of cyclic AMP during gradient development caused a greater and qualitatively different attraction. Attraction in spatial gradients was only transient, in that populations eventually developed a random distribution when confined to a narrow territory. Populations never accumulated nor lost their random distribution even in extremely steep spatial gradients. Attraction in spatial gradients was inducible only in spreading populations, not randomly distributed ones. Thus, spatial gradients effect biased-random locomotion: i.e., chemokinesis without adaptation. Cells cannot read gradients; the reaction of the cells is stochastic. Spatial gradients do not cause chemotaxis, which probably requires a sharp stimulant concentration increase (a temporal gradient) as a pulse or impulse. The results also bear on concepts of how embryonic cells might be able to decipher the positional information in a morphogen spatial gradient during development.

Cellular pattern formation: Dictyostelium discoideum as a system for a biochemical approach

The cellular slime mold Dityostelium discoideum offers a convanient system in which to investigate the biochemical events that underlie the formation of two different cell types during development. Both morphological and biochemical evidence suggest that cyclic AMP plays a key role in the patterning of prestalk and prespore cells in this organism. The antagonistic distribution of adenylate cyclase and phosphodiesterase offers the potential for establishing a gradient of cyclic AMP across the two cell types.

Adenosine 3',5'-monophosphate waves in Dictyostelium discoideum: a demonstration by isotope dilution--fluorography.

The distribution of adenosine 3',5'-monophosphate (cyclic AMP) in fields of aggregating amoebae of Dictyostelium discoideum was examined by a novel isotope dilution-fluorographic technique. Cellular cyclic AMP was visualized by its competition with exogenous 3H-labeled cyclic AMP for high-affinity binding sites on protein kinase immobilized on a Millipore filter used to blot the monolayer. The cyclic AMP was distributed in spiral or concentric circular wave patterns which centered on the foci of the aggregations. These patterns were correlated with those of cell shape change that propagate through the monolayers: cells in regions of high concentrations of cyclic AMP were elongated (presumably moving up a cyclic AMP gradient), whereas those in regions of low cyclic AMP concentrations were randomly directed. The highest cyclic AMP concentrations were about 10(-6)M. The widths of the regions of elevated cyclic AMP were about 0.3 to 1 millimeter which, assuming a wave velocity of 300 micrometers per minute, suggests that a cell signals for about 1 to 3 minutes. These observations support the hypothesis that the aggregation process in Dictyostelium is mediated by the periodic relay of cyclic AMP signals and suggest a simple scheme for the dynamics of the aggregation process.

Extracellular ATP, ecto-ATPase and calcium influx in Dictyostelium discoideum cells

There is evidence that a protein kinase is present on the surface of Dictyostelium discoideum cells [l-3] and is capable of phosphorylating certain plasma membrane proteins [3]. Such phosphorylation may be involved in membrane permeability and transport, cell movement, and the activation of cyclic AMP receptors which occurs during early differentiation [4]. External ATP induces cell aggregation, decreases the chemotactic sensitivity of cells to a cyclic AMP gradient and inhibits intercellular contact sites outside the aggregation center [ 1,5]. We found that ATP (0.1-0.8 PM produced at 1.5 X lo7 cells/ml) is present extracellularly in cell suspensions. This reflected a steady state concentration since a Mg”-dependent ecto-ATPase was continually hydrolyzing extracellular ATP. The ectoATPase was inhibited by suramin and I-ethyl-3(3-dimethyl-aminopropyl)carbodiimide (EDAC), but not by oligomycin or ouabain. Ecto-ADPase, -AMPase and nucleotide diphosphokinase activities were also detected. Calcium influx was inhibited by suramin, l-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide (EDAC), KCN, dinitrophenol, carbonyl-mchlorophenylhydrazone (CCCP), adenyl imidodiphosphate, adenyl methylene diphosphate, and by adding hexokinase or apyrase to split extracellular ATP. Addition of ATP (optimum level 10e6 M) to cell suspensions stimulated calcium influx and partially overcame the KCN-induced inhibition of this influx. Glucose uptake was not affected by suramin or apyrase.

Theoretical analyses of the functioning of the high- and low- K m cyclic nucleotide phosphodiesterases in the regulation of the concentration of adenosine 3′,5′-cyclic monophosphate in animal cells

The concentration of cyclic AMP in a cell is determined by the activities of three enzymes: adenylate cyclase; low- K m phosphodiesterase, and high- K m phosphodiesterase. Analysis of the properties of this system has been undertaken. When the system is treated as three soluble enzymes, the steady state concentration of cyclic AMP in the cell is seen to be relatively insensitive to the activity of high- K m diesterase, which is present in larger amounts than can be readily explained. By numerical simulation of a more realistic model which treats adenylate cyclase and low- K m diesterase as enzymes bound to the plasma membrane, it is demonstrated that the system can exhibit greater flexibility. The content of high- K m diesterase is shown to be sufficient to generate a concentration gradient of cyclic AMP in a cell where adenylate cyclase is on the plasma membrane. This lowers the steady state concentration of cyclic AMP. Although it is normally ineffective at controlling the cyclic AMP concentration near the membrane, the high- K m enzyme can be as effective as the low- K m diesterase at regulating the level further inside the cell. The rate of approach to a new steady state after modulation of cyclase activity is also increased by high- K m diesterase. Although only a single pool of cyclic AMP is assumed, differences are predicted between cyclic-AMP dependent processes at the plasma membrane compared with the interior of the cell. On or near the membrane, modulation of only the cyclase or low- K m diesterase can exert control on the process, and the rate of response will be faster than the rate of change of the cellular content of cyclic AMP. Nervous tissue may be an exception, where the high- K m diesterase does control the concentration near the membrane effectively.

Distribution of Cyclic Nucleotides in the Organ of Corti

Recent developments in radioimmunoassay technology have made possible measurements of cyclic nucleotides in individual specimens of the organ of Corti and its subdivisions. Steep longitudinal and transverse gradients of glycogen are known to exist in the organ of Corti of the guinea pig, with preferential accumulation in the outer hair cells of the apical turns. However, no significant longitudinal gradient of cyclic AMP was detectable in the organ of Corti, and the concentration of the compound was found to be nearly equal in the inner and outer hair cell layers. This is indirect evidence against the concept that cyclic AMP plays a role as "second messenger" in the control of glycogen metabolism of the organ of Corti. By contrast, the concentration of cyclic GMP was found to be consistently higher in the inner layer than in the outer layer of the organ of Corti, and to increase significantly in basal direction. This trend is remarkably similar to the distribution patterns of acetylcholinesterase, which may be considered as indirect evidence in favor of a possible role of cyclic GMP in the mediation of cholinergic effects.

Cyclic AMP, Cyclic AMP Phosphodiesterase, and the Duration of the Interphase in Dictyostelium discoideum

Summary After feeding has ended, amoebae of the cellular slime mold, Dictyostelium discoideum, enter a quiescent period (the interphase) before aggregation. Many characteristics required for aggregation develop in the amoebae during the interphase period, and the length of the interphase is affected by numerous factors including the density of the developing cell population. It was previously shown that the fewer the cells present, the longer the interphase [12, 13]. The present study is an investigation of the cell density dependence of the length of the interphase. It is shown that the interphase can be shortened for cell populations of low density by reducing the external cyclic AMP levels surrounding the amoebae. The same effect is obtained whether the cyclic AMP level is reduced by dilution into salt solution below the support medium, by absorption on to charcoal, or by destruction with the enzyme phosphodiesterase. Additional cyclic AMP in the support medium lengthens the interphase. Measurements of the cyclic AMP phophodiesterase levels produced by amoebae show that, under the condition of a reduced external cyclic AMP level, populations of low cell density not only aggregate earlier, but also aggregate at a lower level of cell-bound phosphodiesterase. At the time of aggregation, external cyclic AMP levels and cell-bound phosphodiesterase levels are seen to reach a specific ratio for lower cell densities. The relationship of cyclic AMP and cell-bound phosphodiesterase at aggregation is interpreted as important in the detection of a cyclic AMP gradient by the receptor system on the surface of the amoebae. It is suggested that low cell densities require a longer period of time to achieve the appropriate relationship between external cyclic AMP and cell-bound phosphodiesterase and therefore have a longer interphase.

Cyclic AMP gradient in migrating pseudoplasmodia of the cellular slime mold Dictyostelium discoideum.

Several lines of experimental evidence suggest that an anterior-posterior gradient of cyclic AMP exists in migrating pseudoplasmodia of the cellular slime mold Dictyostelium discoideum, and that this gradient may be responsible for control of the proportions of stalk and spore cells that form during culmination. In experiments reported here, the amounts of cyclic AMP present in the anterior and posterior portions of pseudoplasmodia were measured using a protein binding assay, and the levels obtained normalized to protein and DNA. For a variety of pseudoplasmodia migration conditions examined, the anterior portion was found to contain cyclic AMP concentrations 40 to 70% higher than the posterior portions.

Effects of progesterone and EDTA on cyclic AMP and phosphodiesterase in Dictyostelium discoideum

WHEN Dictyostelium discoideum amoebae are nutritionally deprived, they undergo a developmental programme which results in the formation of fruiting bodies composed of spore and stalk cells. Early events in this cycle are marked by the aggregation of previously individual amoebae. During this process they become elongated, and establish specific intercellular contacts, resulting in the formation of cell streams1. Orientation of cell movement during this period is thought to be directed by an external gradient of cyclic AMP (ref. 2). This compound elicits a chemotactic response from starved cells but not from growing cells3,4 and can induce a period of coordinated movement in nutritionally deprived cells5. A phosphodiesterase is also excreted by aggregating cells6,7 activity of which modulates the cyclic AMP gradient. Thus, a general model for communication between cells during aggregation is available. Little is known, however, about the regulation of metabolic events which result in the differentiation of growth phase cells into aggregation-competent, cyclic AMP-sensitive cells, and in particular, the influence of cyclic AMP on these processes. Since in other eukaryotes, changes in internal cyclic AMP are thought to mediate the actions of a variety of hormones, and regulate cell growth and development, we investigated the possible intracellular role of this nucleotide in D. discoideum.

Adenosine 3′, 5′-cyclic monophosphate in higher plants: Assay, distribution and age-dependency

Adenosine 3′,5′-cyclic monophosphate does occur in higher plants and is widely distributed both in monocots and dicots. Its level varies in roots, leaves and buds, and changes with age. Wide differences exist between annual plants perennial woody trees. A concentration gradient of cyclic AMP exists from the apex to the roots.

Directional effects of cyclic AMP on platelets.

1. Platelets undergo a spreading reaction in a microscope slide—coverslip preparation. 2. If a gradient of cyclic AMP is generated in such a preparation a large fraction of the spread platelets are oriented with their veils into the gradient.

Cyclic AMP, pattern formation and movement in the slime mould, Dictyostelium discoideum

It is important to determine whether a gradient of cyclic AMP exists along the migrating slime mould grex because such a gradient might be involved in pattern formation and the polarity of grex movement. Biochemical measurements provided results which could be consistent with Bonner's suggestion that 50% of the cyclic-AMP produced by the grex is produced by the anterior one-tenth. However, chemotactically sensitive cells cannot detect a gradient of cyclic-AMP emission along the grex. Migrating grex cells themselves are not chemotactically sensitive to cyclic-AMP. It seems unlikely that chemotaxis is involved in controlling the polarity of grex movement.