Regulation of protein synthesis during the preaggregative period of Dictyostelium discoideum development: involvement of close cell associations and cAMP

The preaggregative period of Dictyostelium discoideum is composed of two rate-limiting components which exhibit dramatic differences in either their dependency upon, or sensitivity to, close cell-cell associations, inhibitors of protein synthesis, temperature, and pH. The first component comprises the initial 4.5 hr and the second component the last 2.5 hr of the preaggregative period. By pulse-labeling cells with [35S]methionine, separating polypeptides by 2D-PAGE, and semiquantitatively comparing the rates of synthesis of 778 individual polypeptides by fluorography, the following results were obtained: a detailed program of protein synthesis accompanies the preaggregative (0-7 hr) and aggregative (7-10.5 hr) periods of development; this includes significant decreases in the rate of synthesis of 93 polypeptides synthesized during vegetative growth and significant increases in the rate of synthesis of 74 polypeptides either undetectable or synthesized at relatively low rates during vegetative growth; 35 polypeptides are transiently synthesized at different times during the preaggregative and aggregative periods; two peaks of activity are clearly defined for both increases and decreases; these peaks correlate temporally with the first and second rate-limiting components of the preaggregative period; the majority of changes (74%) which occur during the first rate-limiting component will occur in the absence of close cell-cell associations, but the majority (66%) which normally occur during the second rate-limiting component do not occur in the absence of close cell-cell associations; a high concentration of cAMP in the medium of continuous suspension cultures does not stimulate most of the changes which are dependent upon close cell-cell associations; even though cAMP stimulates progress through the second rate-limiting component in suspension cultures first allowed to associate for 4.5 hr ("competent" cells) prior to disaggregation it still does not stimulate most of the changes which are dependent upon close cell-cell associations; and synthesis of only 3 out of 778 polypeptides appears to be stimulated by addition of exogenous cAMP, and only in resuspended cultures of "competent" cells. The prominent role of close cell-cell association and the surprisingly minor effect of cAMP in the regulation of the program of protein synthesis accompanying the preaggregative and aggregative periods of Dictyostelium are discussed, especially as they relate to the effect of cAMP on protein synthesis in suspended cultures of postaggregative cells.     

Dictyostelium discoideum lipids modulate cell-cell cohesion and cyclic AMP signaling.

During Dictyostelium discoideum development, cell-cell communication is mediated through cyclic AMP (cAMP)-induced cAMP synthesis and secretion (cAMP signaling) and cell-cell contact. Cell-cell contact elicits cAMP secretion and modulates the magnitude of a subsequent cAMP signaling response (D. R. Fontana and P. L. Price, Differentiation 41:184-192, 1989), demonstrating that cell-cell contact and cAMP signaling are not independent events. To identify components involved in the contact-mediated modulation of cAMP signaling, amoebal membranes were added to aggregation-competent amoebae in suspension. The membranes from aggregation-competent amoebae inhibited cAMP signaling at all concentrations tested, while the membranes from vegetative amoebae exhibited a concentration-dependent enhancement or inhibition of cAMP signaling. Membrane lipids inhibited cAMP signaling at all concentrations tested. The lipids abolished cAMP signaling by blocking cAMP-induced adenylyl cyclase activation. The membrane lipids also inhibited amoeba-amoeba cohesion at concentrations comparable to those which inhibited cAMP signaling. The phospholipids and neutral lipids decreased cohesion and inhibited the cAMP signaling response. The glycolipid/sulfolipid fraction enhanced cohesion and cAMP signaling. Caffeine, a known inhibitor of cAMP-induced adenylyl cyclase activation, inhibited amoeba-amoeba cohesion. These studies demonstrate that endogenous lipids are capable of modulating amoeba-amoeba cohesion and cAMP-induced activation of the adenylyl cyclase. These results suggest that cohesion may modulate cAMP-induced adenylyl cyclase activation. Because the complete elimination of cohesion is accompanied by the complete elimination of cAMP signaling, these results further suggest that cohesion may be necessary for cAMP-induced adenylyl cyclase activation in D. discoideum.     

A cytosolic cyclic AMP-dependent protein kinase in Dictyostelium discoideum. II. Developmental regulation

The cAMP-dependent protein kinase of the cellular slime mold, Dictyostelium discoideum, is developmentally regulated; there is an approximately 4-fold increase in activity during development. The incorporation of [3H]leucine into the enzyme demonstrates that there is de novo synthesis of the cAMP-dependent protein kinase. The activities of the catalytic and regulatory subunits increase in parallel. The maximal rate of increase of cAMP-dependent protein kinase activity precedes "tip" formation, a stage of development characterized by a sharp increase in mRNA complexity. The high level of cAMP-dependent protein kinase activity, attained at this stage of development, persists when aggregates are dispersed and the amoebae are kept in suspension without added cAMP. The synthesis of the developmentally regulated mRNAs under these conditions is dependent on exogenous cAMP. The increase in cAMP-dependent protein kinase activity during development does not require sustained cell-cell contact insofar as it occurs in single cell suspensions of amoebae. Furthermore, the increase does not require exogenous cAMP, although added cAMP stimulates the synthesis of the enzyme to a level higher than that found, when cAMP is not added. These observations support the hypothesis that in D. discoideum cAMP-dependent protein kinase mediates the effects of cAMP on development.     

Synthesis and stability of developmentally regulated dictyostelium mRNAs are affected by cell-cell contact and cAMP

Postaggregation Dictyostelium discoideum cells contain 3000 mRNA species that are absent from preaggregation cells; these aggregation-dependent sequences comprise 30% of the mass of mRNA in these cells. We show that the synthesis and stability of these regulated mRNA sequences are affected by both cell-cell contact and cAMP. Three independent assays are used to quantitate these mRNAs: in vitro translation followed by two-dimensional gel analysis of the protein products; hybridization of gel-separated RNAs to cloned DNAs; and hybridization of mRNA to a cDNA probe specific for the population of regulated sequences. In postag-gregation cells, the half-life of both the developmentally regulated mRNAs and the constitutive mRNAs present throughout growth and differentiation is the same—about 4 hr. Following disaggregation, all of the late mRNA sequences are degraded and decay with a half-life of 25 to 45 min. The constitutive species are unaffected; 2.5 hr after disaggregation, the ratio of late to constitutive mRNAs is about 6% that of normal plated cells. Addition of cAMP to cells that have been disaggregated for 2.5 hr (or longer) restores the level of most late mRNAs within 3 hr. We conclude that cAMP stimulates the synthesis of these mRNAs and may also act to stabilize them in the cytoplasm. This effect of cAMP is dependent on the cells having been in contact with other cells; cAMP has no effect on the levels of mRNA in suspension-starved, aggregation-competent cells that have never formed cell-cell aggregates.     

Induction and modulation of cell-type-specific gene expression in Dictyostelium

We have identified genes that are expressed preferentially in either prestalk or prespore cells in Dictyostelium. The prestalk mRNAs are detectable at 7.5 hr prior to the completion of cell aggregation, while the prespore mRNAs are not detectable until approximately 15 hr of development. Exogenous cAMP in the absence of sustained cell contact is sufficient to induce prestalk-specific gene expression, while multicellularity is required for the induction of prespore-specific genes. A gene expressed equally in both cell types, which has the same developmental kinetics as the prestalk genes, is induced in shaking culture in the absence of either cAMP or stable cell associations. Dissociation of aggregates results in the rapid loss of prespore- and prestalk-specific mRNAs, and these can be induced to reaccumulate with the addition of cAMP. We conclude that there are substantial differences in the timing and requirements for tissue-specific gene expression in Dictyostelium.     

Apparent Positive Cooperativity at a Surface cAMP Receptor in Dictyostelium

In the large species of the cellular slime mold Dictyostelium , cell aggregation is regulated by extracellular cAMP. During aggregation, cAMP is released in pulses from cells in the aggregation centers and these rhythmic signals are propagated through the population by a signal relay system. In addition to triggering the relay response, the pulsatile signals also regulate the chemotactic movement of the cells and early cell differentiation. These different cellular responses to exogenous cAMP are thought to be mediated via cAMP receptors, which appear on the cell surface shortly after starvation.
Using a sensitive assay, the equilibrium binding properties of these receptors were analyzed at low cAMP concentrations. As reported earlier, Scatchard plots of cAMP binding to preaggregative amoebae of D. discoideum strain NP187 in the concentration range 2–500 nM were curvilinear suggesting either receptor heterogeneity or negative cooperative interactions. However, at cAMP concentrations below approximately 1.5 nM, the affinity of the receptors was found to decline as a function of decreasing receptor occupancy. This apparent positive cooperativity was observed with binding sites on crude plasma membranes as well as on intact cells, and it occurred at both 0°C and 22°C. Moreover, apparent positive cooperativity was a property of the receptors on all strains of D. discoideum examined and on one strain of D. purpureum . Unlike preaggregative cells, receptors on postaggregative cells often lacked this property.
The lowest concentration of cAMP pulses that can appreciably stimulate membrane differentiation in strain NP187 was found to be 0.15–1.5 nM. Since similar concentrations of exogenous cAMP have been reported to trigger minimal chemotactic and relay responses in D. discoideum , the apparent positive cooperative behavior of the cAMP receptor might function to generate a steep cellular response threshold.     

The regulation of''early'' enzymes during the development and dedifferentiation of Dictyostelium discoideum

The specific activities of the enzymes alpha-mannosidase and N-acetylglucosaminidase increase immediately after the initiation of the development of bacterially grown cell cultures of Dictyostelium discoideum. The regulation of these two enzymes was found to be dissociable in the developmental timer mutant, FM-1, which aggregates 4.5 h earlier than wild-type cells due to the absence of the first rate-limiting component of the preaggregative period. The increase in alpha-mannosidase activity occurs in the absence of the first rate-limiting component, but the increase in N-acetylglucosaminidase activity does not. These results indicate the following: (1) the increase in the specific activity of alpha-mannosidase is not related to the timing of subsequent developmental stages; (2) the increase in the specific activity of N-acetylglucosaminidase is not necessary for the subsequent developmental program; and (3) either the increase in the specific activity of N-acetylglucosaminidase is dependent upon progress through the first rate-limiting component, or the increase in this enzyme activity and the first rate-limiting component are both dependent upon an early event for which FM-1 is defective. In addition to early development, we monitored the two enzyme activities during dedifferentiation. The results demonstrate that there is no difference between dedifferentiating wild-type cells and dedifferentiation-defective mutant HI-4 cells. Changes in enzyme specific activity accompanying dedifferentiation are dependent upon the composition of the dedifferentiation-inducing media and are consistent with the levels of these enzymes observed in cells growing in the different nutrient media.     

The developmental regulation of L-ornithine decarboxylase in Dictyostelium discoideum and its induction by cAMP

By the use of an in vivo assay, ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) is shown to be developmentally regulated in Dictyostelium discoideum. High levels of cAMP can induce ornithine decarboxylase activity in preaggregative cells kept in shaking suspension, under similar conditions as where other markers for development can also be induced. This induction by cAMP is solely dependent on the total amount of cAMP to which the cells have been exposed, and not on the manner of cAMP addition. Induction of ornithine decarboxylase activity, when measured in vitro, is caused by both an increase in total enzyme activity and by a proportional increase in activity of the high-affinity form for the cofactor pyridoxal phosphate. When measured in vivo, an additional regulatory mechanism seems to be involved. Kinetic studies with the competitive inhibitor putrescine suggest that in cAMP-stimulated cells the low affinity form of the enzyme may also be active in vivo.     

Reconstitution of Embryo-Like Structures from Sea Urchin Embryo Cells: Distinction Between Cell-Cell and Cell-Substrate Associations

Sea urchin embryos can be dissociated into a suspension of single cells that reconstitute embryo-like structures. When reconstitution is conducted in stationary cultures the first step is attachment of the cells to the culture plate, which requires calcium and metabolic energy but not protein synthesis. We have found that protease treated cells form cell-cell associations in stationary cultures without attaching to the culture plates, and that cell-plate attachments are unaffected by inhibition of protein synthesis. These data suggest that cell surface proteins are needed for cell-plate attachment and that these proteins are present on freshly dissociated cells. We also demonstrated that butanol extracted cells attach to the plates, but do not form functional cell-cell associations unless the butanol extracted material is restored to them. We conclude that sea urchin embryo cells contain two classes of attachment components. The first class functions in the cell-plate attachments, is protease sensitive, and not extracted by butanol; the second class is necessary for cell-cell associations, is protease insensitive, and extracted by butanol. Since protease treated cells reconstitute embryo-like structures without attaching to the culture plates, only the second class of attachment components is necessary for embryo reconstitution.     

Characterization of genes which are deactivated upon the onset of development in Dictyostelium discoideum

We have identified and begun characterizations of the differential expression of 15 genes whose corresponding mRNA levels decrease during the preaggregative period of the developmental program of Dictyostelium discoideum. Upon the onset of development, the mRNAs decrease from 5- to 1000-fold over the first 8 hr. The rates of loss of each mRNA were similar to one another but distinct, and the decreases were dependent on progress through the developmental program. One exception to this dependency was observed, and the decrease in this mRNA was dependent on the absolute time after initiation of development instead of progress through development. With two exceptions, the decreases in mRNA levels were dependent on developmental conditions and were not seen when cells were shaken in starvation buffer. When the polysomal distributions of each species were examined, three classes were found: most showed no significant shifts off of polysomes upon initiation of development, two were characterized by a 20% shift to nonpolysomal RNA fractions upon development, and two gave a 40-50% shift. Collectively, these characterizations reveal differences in behavior which suggest that deactivation of genes upon initiation of development in Dictyostelium involves more than one regulatory pathway.     

Acquisition process of differentiation competence in Dictyostelium discoideum

It was previously shown [K. Okamoto, J. Gen. Microbiol. 127, 301 (1981)] that Dictyostelium discoideum cells dissociated from early aggregates, but not aggregation competent cells obtained in a suspension culture, undergo prespore differentiation, when transferred into a medium containing glucose, albumin, and cAMP. Therefore, the former, but not the latter, is considered to have been acquired "differentiation competence." In the present work, the requirements for cells to acquire the differentiation competence are investigated with D. discoideum NC4 strain. On solid substratum, the incubation above a threshold density is absolutely required for this process, while cell aggregation itself is not essential. In suspension cultures, the competence is acquired only under hypertonic conditions. Inhibition of protein synthesis or depletion of cAMP does not affect the acquisition process of the competence. The requirement of hypertonic treatment was also investigated with several other D. discoideum strains.     

Noncoordinate regulation of de novo synthesis of cytochrome P-450 cholesterol side-chain cleavage and cytochrome P-450 17 alpha-hydroxylase/C17-20 lyase in mouse Leydig cell cultures: relation to steroid production

The role of cAMP in the regulation of the amount and synthesis of cytochrome P-450 cholesterol side-chain cleavage (P-450scc) and cytochrome P-450 17 alpha-hydroxylase/C17-20 lyase (P-450(17 alpha) was investigated in mouse Leydig cell cultures. In the absence of cAMP, the amount of immunoreactive P-450(17 alpha) decreased to less than 5% by day 4 and was undetectable between days 7 and 11. In contrast, the amount of immunoreactive P-450scc remained relatively constant throughout the same period. Treatment of Leydig cell cultures for 4 days with 0.05 mM 8-bromo-cAMP initiated on day 7 increased the amount of P-450(17 alpha) with relatively little effect on the amount of P-450scc. The rate of de novo synthesis of each of the P-450 enzymes was studied by determining [35S]methionine incorporation into newly synthesized protein. In the absence of cAMP, de novo synthesis of P-450(17 alpha) ceased while the rate of de novo synthesis of P-450scc increased with time in culture between days 2 and 11. Treatment with cAMP initiated on day 7 of culture caused a time-dependent increase in the rate of de novo synthesis of P-450(17 alpha) on days 9 and 11 equivalent to 40% and 60%, respectively, of that observed in freshly isolated Leydig cells. The rate of de novo synthesis of P-450scc was increased 2-fold relative to untreated cultures on days 9 and 11. De novo synthesis of P-450(17 alpha) ceased when cAMP was removed on day 11 and restored when cAMP was added again on day 13 of culture.(ABSTRACT TRUNCATED AT 250 WORDS)     

Different molecular mechanisms for cAMP regulation of gene expression during Dictyostelium development

Alterations in cAMP concentrations have been implicated in developmentally regulated gene expression in Dictyostelium. Using a variety of culture conditions to control the metabolism of cAMP during cytodifferentiation, I have examined the role of the cyclic nucleotide in development. Conditions which allow intracellular synthesis of cAMP promote the normal developmental repression of gene M4-1 by a mechanism which is completely independent of the formation of multicellular aggregates. If, however, cells are inhibited in their ability to activate adenylate cyclase and, thus, intracellular cAMP signaling, they prove unable to repress M4-1, even in the presence of exogenous cAMP. In contrast, expression of genes which exhibit maximal activity after aggregate formation depends upon accumulation of extracellular cAMP. Inhibition of intracellular cAMP signaling does not prevent the expression of these genes if cultures are simultaneously exposed to high levels of exogenously added extracellular cAMP. These results indicate that there are at least two independent mechanisms involved in the developmental regulation of gene expression by cAMP in Dictyostelium. I discuss plausible molecular mechanisms through which cAMP might alter gene expression.     

Disaggregation-induced changes of developmentally regulated proteins in Dictyostelium discoideum

By means of two-dimensional gel electrophoresis, we analyzed proteins present in a slug-shaped tissue mass of D. discoideum and examined the changes in their amounts after disaggregation of the slugs. Of approximately one hundred polypeptides, six were found to decrease in amount after disaggregation. The decreases of four polypeptides were inhibited by the presence of 1 mM cAMP or 250 micrograms/ml cycloheximide. The decreases of the two other proteins were not suppressed by cAMP or cycloheximide. The patterns of proteins present in vegetative and aggregative cells were also examined. None of the six proteins which showed a decrease after slug disaggregation was found in vegetative or preaggregative cells. These results indicate that both synthesis and degradation of these proteins are controlled by cell-cell contact.     

Regulation of gene expression by the intracellular second messengers IP3 and diacylglycerol

In Dictyostelium, extracellular cAMP interacts specifically with cell-surface receptors to promote the accumulation of a variety of intracellular second messengers, such as 3'-5' cyclic adenosine monophosphate (cAMP) and 1,4,5 inositol trisphosphate (IP3). We and others have shown that activation of the cell-surface cAMP receptor can also modulate the expression of the Dictyostelium genome during development. In at least one instance, synthesis of intracellular cAMP is required for appropriate gene regulation. However, the induction of most cAMP-dependent gene expression can occur in the absence of receptor-mediated activation of adenylate cyclase and a consequent accumulation of intracellular cAMP. These results suggest that other intracellular second messengers produced in response to receptor activation may potentially act as signal transducers to modulate gene expression during development. In vertebrate cells, IP3 and diacylglycerol (DAG) are intracellular activators of specific protein kinases; they are produced in equimolar amounts by cleavage of phosphoinositol bisphosphate after a receptor-mediated activation of a membrane-bound phosphodiesterase. IP3 and, thus, by inference, diacyl-glycerol are synthesized in Dictyostelium as a response to cAMP interacting with its cell-surface receptor. Using defined conditions to inhibit the accumulation of extracellular cAMP, we have examined the effects of these compounds on the expression of genes that require cAMP for their maximal expression. Our results suggest that intracellular IP3 and DAG may in part mediate the action of extracellular cAMP on the expression of the Dictyostelium genome.     

Cell-cell contact and cAMP regulate the expression of a UDP glucose pyrophosphorylase gene of Dictyostelium discoideum

UDP glucose pyrophosphorylase (UDPGP) (EC.2.7.7.9) is a developmentally regulated enzyme of Dictyostelium discoideum. Two polypeptides of UDPGP are translated from Dictyostelium mRNA. Recently we isolated a cDNA clone which encodes one of the UDPGP polypeptides (B. R. Fishel, J. A. Ragheb, A. Rajkovic, B. Haribabu, C. W. Schweinfest, and R. P. Dottin (1985). Dev. Biol. 110, 369-381). By hybridization with the cDNA and by in vitro translation and immunoprecipitation, we examined the effect of cell-cell contact and cAMP on the regulation of UDPGP expression. Disaggregation of slugs resulted in a rapid loss of UDPGP mRNA. Addition of cAMP to these cells resulted in increased levels of UDPGP mRNA, though not to the same extent as seen during normal development. The two UDPGP polypeptides observed in vitro are coordinately regulated. Unaggregated cells, starved and shaken rapidly in suspension, did not show UDPGP mRNA accumulation. However, addition of cAMP to these cells caused UDPGP induction, suggesting that the requirement for cell-cell contact could be bypassed in part by cAMP addition.     

Unsaturated free fatty acids regulate cAMP relay and cell-cell adhesion duringDictyostelium discoideum aggregation

Summary Cell-cell adhesion and cAMP-stimulated cAMP production (cAMP relay) regulate the aggregation that occurs early in theDictyostelium discoideum developmental cycle. Increasing the concentration of neutral lipids inD. discoideum membranes inhibits both cell-cell adhesion and cAMP relay. Fractionation experiments revealed that it was the free fatty acids, one group of the neutral lipids, that inhibited both cAMP relay and cell-cell adhesion. Work with commercially-available free fatty acids demonstrated that the addition of saturated free fatty acids, palmitic acid and stearic acid, did not alter cell-cell adhesion or cAMP relay. The addition of unsaturated free fatty acids inhibited both cAMP relay and cell-cell adhesion in a dose-dependent, saturable manner. To test the physiological significance of these observations, the concentrations of endogenous unsaturated fatty acids were modified by altering theD. discoideum diet. Decreasing the amount of fatty acids 18 carbons long with 2 double bonds enhanced cAMP relay and cell-cell adhesion. These results suggest that fatty acids may be important regulators of cAMP responsiveness and cell-cell adhesion duringD. discoideum aggregation, and therefore may play important roles in development.     

Relationships between cell-cell interactions,cAMP, and gene expression in a developmental mutant ofDictyostelium discoideum

Previous work has led us to propose that close cell-cell associations duringD. discoideum development serve as a signal to deactivate expression of discoidin I mRNA, and that intracellular cAMP serves as a mediator of this regulatory pathway. This model is based in part on the failure of a morphogenetic mutant, EB-21, to deactivate discoidin I expression under conditions where these cells fail to acquire cell-cell cohesiveness and hence remain as single cells, unlike the wild type strain which forms multicellular aggregates. Here we show that the failure of EB-21 to express specific cohesiveness depends on developmental conditions, and that under conditions where close cell-cell associations are allowed to form, discoidin I mRNA expression is deactivated normally. Furthermore, in both wild type and EB-21 there is a close correlation between formation close cell-cell associations and elevation of intracellular cAMP under different developmental conditions. Additional analyses of the biological behavior of EB-21 indicate that it acquires a normal cAMP chemotactic signal-response system, and that the morphogenetic defect cannot be corrected by co-development with wild type cells. The results are discussed in terms of possible relationships between cell-cell interactions, cAMP metabolism, and developmental gene expression in this organism.Dedicated to Dr. E. M. Shooter and Dr. S. Varon as part of a special issue (Neurochemical Research, Vol. 12, No. 10, 1987).