Changes in activity of enzymes associated with prespore differentiation in a suspension culture of Dictyostelium discoideum

It has been shown (Okamoto, K. (1981) J. Gen. Microbiol. in the press) that Dictyostelim discoideum cells dissociated at early aggregation can differentiate into prespore cells in a suspension containing glucose, albumin, EDTA and cyclic AMP. Strict requirement of cyclic AMP in this process has also been demonstrated. In the present paper, changes in activity of eight developmentally regulated enzymes were examined in this culture system and compared to those occuring in the normal course of development on the solid substratum. The results show that (a) formation in this medium is not accompanied by increases in activity of UDPglucose pyrophosphorylase and trehalose phosphate synthetase, unlike the case of the normal development, (b) among the enzymes examined, only UDPgalactose: polysaccharide galactosyl transferase can be regarded as a specific marker of the prespore formation, and (c) development in this system does not proceed beyond the slug stage of the normal development, in the case of a wild-type strain NC4.     

Inhibition of the development of Dictyostelium discoideum by isolated plasma membranes

The life cycle of Dictyostelium discoideum can be divided into two mutually exclusive phases: growth and development. A distinguishing characteristic of the two phases is the absence of intercellular communication during vegative growth, and the many forms of such interaction during development. We have investigated the role of the cell surface membrane during the aggregation and development of this organism. We have asked the question: Are there molecules on the cell surface which are necessary for aggregation, and if so, can they be isolated in a biologically active membrane preparation? Further, when do these molecules appear during normal development, and does the interaction between two neighboring cell surfaces signal the cell or affect their subsequent development in any way? We have been able to isolate a partially purified plasma membrane fraction which is capable of specifically blocking the aggregation of other cells. Additional characterization of this preparation suggests that isolated aggregation phase membranes display a new, or newly exposed, heat-stable component which is capable of interacting with vegetative cells in such a way as to halt development.     

Developmental regulation of a stalk cell differentiation-inducing factor in Dictyostelium discoideum

Previous work has shown that cells developing at high density release a low-molecular-weight factor that can induce isolated Dictyostelium discoideum amoebae of strain V12M2 to differentiate into stalk cells in the presence of cyclic AMP. We now show that this differentiation-inducing factor, called DIF, can be extracted from cells during normal development and that its production is strongly developmentally regulated. DIF is not detectable in vegetative cells but rises dramatically after aggregation to reach a peak during slug migration. DIF levels are very low in two mutants defective in aggregation. The postaggregative synthesis of DIF is stimulated by the addition of extracellular cyclic AMP. We propose that DIF is a morphogen controlling prestalk cell differentiation.     

Regulation and secretion of early developmentally controlled enzymes during axenic growth in Dictyostelium discoideum

The four earliest developmentally controlled enzymes in the cellular slime mold, Dictyostelium discoideum, accumulate during axenic growth in rich media. We have shown that at low cell titers the specific activities of N-acetylglucosaminidase, α-mannosidase, leucine aminopeptidase, and alanine transaminase are each at very low or vegetative levels comparable to amoebae which have been grown on bacteria as the food source. During the exponential phase of growth all four enzymes accumulate dramatically reaching cellular specific activities at least as high as during development. The magnitude of this accumulation is influenced by alterations in the growth medium. We suggest that these results, combined with those of prior investigations, indicate that a restricted segment of early development is initiated during axenic growth. This means that growth and early development are not mutually exclusive events in this organism. The secretion of lysosomal enzymes is also affected by the composition of the growth media. In all media, including growth in bacterial suspensions, lysosomal enzymes are secreted in significant quantities. There is a correspondence in the effects of media composition on the secretion of these enzymes and on the regulation of developmentally controlled enzymes during axenic growth. The secretion of lysosomal enzymes that are not developmentally regulated is affected in these media, suggesting that the regulation and secretion of these enzymes are under separate control. It is clear that studies of the regulation of lysosomal enzymes in this organism must take into account the secretion of the enzymes as well as their cellular specific activities to properly reflect levels of gene expression.     

In vivo sulfation of the contact site A glycoprotein of Dictyostelium discoideum

During the development of Dictyostelium discoideum from the growth phase to the aggregation stage, a glycoprotein with an apparent mol. wt. of 80 kd is known to be expressed on the cell surface. This glycoprotein, referred to as contact site A, has been implicated in the formation of species-specific, EDTA-stable contacts of aggregating cells. When developing cells were labeled in vivo with [³⁵S]sulfate, the 80-kd glycoprotein was found to be the most prominently sulfated protein. Another strongly sulfated protein had an apparent mol. wt. of 130 kd and was, like the 80-kd glycoprotein, developmentally regulated and associated with the particulate fraction of the cells. The [³⁵S]sulfate incorporated into the 80-kd and 130-kd proteins was not present as tyrosine-O-sulfate, a modified amino acid found in many proteins of mammalian cells. D. discoideum cells incubated with [³⁵S]sulfate in the presence of tunicamycin, an inhibitor of N-glycosylation, produced a 66-kd protein that reacted with monoclonal antibodies raised against the 80-kd glycoprotein, but no longer contained [³⁵S]sulfate. These results suggest that sulfation of the 80-kd glycoprotein occurred on carbohydrate residues. The possible importance of sulfation for a role of the 80-kd glycoprotein in cell adhesion is discussed.     

The alpha-glucosidases of Dictyostelium discoideum. II. Developmental regulation and cellular localization

Four isozymes of α-glucosidase in Dictyostelium discoideum have been identified and some of their enzymatic and physical properties characterized (R. H. Borts and R. L. Dimond, 1981, Develop. Biol.87, 176–184). In this report the cellular localization and developmental regulation of three of these isozymes are determined. α-Glucosidase-1 is the major isozyme of vegetative amoebae. It is lysosomally localized and secreted from the cell under certain conditions. It has an acidic pH optimum and carries the common antigenic determinant found on all lysosomal enzymes in this organism. The specific activity of this isozyme begins to decrease within a few hours after the initiation of development and is no longer detectable in the mature fruiting body. α-Glucosidase-2 has a neutral pH optimum and is neither lysosomal nor secreted. Rather it is membrane bound and is possibly located on the cisternal side of microsomal vesicles. This isozyme does not possess the common antigenic determinant. α-Glucosidase-2 comprises 20–40% of the total α-glucosidase activity of the vegetative cell. Its specific activity increases threefold during development. This isozyme appears to be developmentally controlled since it fails to accumulate in aggregation deficient mutants. Its accumulation is also dependent upon continued protein synthesis. α-Glucosidase-4, like α-glucosidase-1, has an acidic pH optimum. It does not appear to be lysosomally localized nor membrane bound. Approximately 30% of the activity is precipitable by antibody against the common antigenic determinant indicating that it is less highly modified or fewer molecules are modified. The isozyme is undetectable during vegetative growth and does not begin to accumulate until late aggregation. Activity peaks in mature fruiting bodies where it is the predominant acidic α-glucosidase activity. Accumulation of α-glucosidase-4 is blocked in morphologically deficient mutants and by inhibitors of protein synthesis.     

Arrestins function in cAR1 GPCR-mediated signaling and cAR1 internalization in the development of Dictyostelium discoideum

Oscillation of chemical signals is a common biological phenomenon, but its regulation is poorly understood. At the aggregation stage of Dictyostelium discoideum development, the chemoattractant cAMP is synthesized and released at 6-min intervals, directing cell migration. Although the G protein–coupled cAMP receptor cAR1 and ERK2 are both implicated in regulating the oscillation, the signaling circuit remains unknown. Here we report that D. discoideum arrestins regulate the frequency of cAMP oscillation and may link cAR1 signaling to oscillatory ERK2 activity. Cells lacking arrestins (adcB⁻C⁻) display cAMP oscillations during the aggregation stage that are twice as frequent as for wild- type cells. The adcB⁻C⁻ cells also have a shorter period of transient ERK2 activity and precociously reactivate ERK2 in response to cAMP stimulation. We show that arrestin domain–containing protein C (AdcC) associates with ERK2 and that activation of cAR1 promotes the transient membrane recruitment of AdcC and interaction with cAR1, indicating that arrestins function in cAR1-controlled periodic ERK2 activation and oscillatory cAMP signaling in the aggregation stage of D. discoideum development. In addition, ligand-induced cAR1 internalization is compromised in adcB⁻C⁻ cells, suggesting that arrestins are involved in elimination of high-affinity cAR1 receptors from cell surface after the aggregation stage of multicellular development.     

The cell cycle and its relationship to development in Dictyostelium discoideum.

When deprived of exogenous nutrients some amoebas of Dictyostelium discoideum do continue to progress through the cell cycle. There are two distinct periods when mitotic cell division occurs. Labeling studies show that during the first period, which begins at the onset of development and ceases at the first visible signs of aggregation (rippling), only those cells which are beyond a certain point in G2 at the initiation of development divide. The second period of mitotic activity begins at tip formation, reaches maximum activity at the grex stage, and ceases during early culmination. Significantly, examination of the development of amoebas harvested when in the stationary phase of growth (and thus arrested in G2) shows that these cells still undergo mitotic cell division during the second period but do not show any such division during the preaggregation phase. The extent to which increases in cell number can be taken to be indicative of mitotic cell division varies from one culture to another due to the presence of variable numbers of multinucleate cells which become mononucleate during the first 10 hr of development. However, when due allowance has been made for the existence of these cells in axenically growing amoebal populations, our data show that by completion of fruiting body construction there has been a doubling in cell number as a direct result of mitotic cell division. Nuclear DNA synthesis also occurs at two distinct periods during development, these coinciding with the periods of mitotic activity. However, since no more than 35% of the cells have undergone nuclear DNA synthesis by the end of the developmental phase, our results are inconsistent with the conclusion that all cells accumulate at a position in G2 at the time of aggregation. Our results do suggest, however, that mitotic cell division of a fraction of the cells may be an integral part of the developmental phase.     

Regulation of gene expression in Dictyostelium discoideum cells exposed to immobilized carbohydrates

When amoebae of Dictyostelium discoideum develop on gels of polyacrylamide that are derivatized with glucosides, they become capable of aggregation at the same time as cells not exposed to glucosides. However, the aggregation centers and streams of adherent cells formed on immobilized glucosides suddenly disintegrate. The cells repeatedly re-aggregate, but never form tight aggregates as they do on other substrata. Tight aggregates formed in the absence of glucosides disperse after their transfer to glucoside gels, and the cells undergo aggregation-disaggregation cycles. The formation of tight aggregates is correlated with the expression of specific post-aggregative poly(A)⁺ RNAs. These RNAs are not expressed in cells developing on glucoside gels, and the dispersal of tight aggregates on such gels is accompanied by the almost complete loss of these RNAs. A developmentally regulated membrane glycoprotein called contact site A, which is a marker of aggregation-competent cells, is normally expressed on glucoside gels. Cyclic AMP is also produced, indicating that the strong increase of adenylate cyclase activity during the preaggregation phase is not affected. In conclusion, cell contact with immobilized glucosides specifically inhibits postaggregative gene expression and arrests development at the aggregation stage.     

The regulation of cellular slime mold development: a factor causing development of Polysphondylium violaceum aggregation-defective mutants.

aggA mutants of Polysphondylium violaceum develop normally in synergistic mixtures with other aggregation-defective mutants. Cell to cell contact is not necessary for development. A small dialyzable factor(s) produced by wild-type and other aggregation-defective mutants triggers development of aggA mutants. This factor (D factor) is developmentally regulated, appearing early in development and then disappearing. Mutants require D factor until aggregation has just begun and then they can continue even in the absence of added factor. D factor is produced by many, but not all species of cellular slime molds and is developmentally regulated in Dictyostelium discoideum as well as P. violaceum.     

The cyclic AMP control system in the development of Dictyostelium discoideum. I. Cellular dynamics.

A model for the synthesis and release of cyclic AMP in aggregating cells of Dictyostelium discoideum is developed. The model shows transitions from low level steady release of cAMP to excitable pulsatile release and then to autonomous periodic pulsatile release of cAMP as starvation proceeds. Finally, there is a transition to high level continuous release of cAMP. A detailed correspondence is drawn between these transitions and the phenomena that are observed to appear sequentially during the aggregation phase, specifically: cloud formation, relaying competence, autonomous competence, and tip activity. The only assumptions necessary to the model are that there is a autocatalytic mechanism for cAMP synthesis, a negative feedback regulation of cAMP through another variable C, and a source term for C that declines with starvation. By analogy with other systems across the phylogenetic scale, in which cAMP activates catabolic pathways and catabolites depress cAMP levels, C is tentatively identified as some measure of the level of energy-yielding catabolites in the cell and the source term for C, as a measure of the cells stored reserves. Starvation for C induces catabolism of stored reserves S through a rise in cAMP. As S, the source term for C declines, the feedback regulation through C can no longer maintain homeostosis and the control loop may be destabilised by small perturbations, i.e. it becomes excitable. A further decline in S can produce limit cycle oscillations in the catabolite-cAMP feedback loop. As S declines even further, continuous steady release of cAMP may ensue.In addition to incorporating the four developmental transitions observed during the aggregation phase as direct consequences of starvation, the model features a super-exponential emergence of relaying competence, phase shifts and acceleration of development by cAMP pulses, and a decreasing refractory period that becomes less than the period of an autonomous cell. All these features closely parallel experimental findings. Finally, the model suggests further experiments critical to an understanding of the dynamics underlying aggregation.     

Detection and characterisation of NAD(P)H-diaphorase activity in Dictyostelium discoideum cells (Protozoa)

In Dictyostelium discoideum (D. discoideum), compounds generating nitric oxide (NO) inhibit its aggregation and differentiation without altering cyclic guanosine monophosphate (cGMP) production. They do it by preventing initiation of cyclic adenosine monophosphate (cAMP) pulses. Furthermore, these compounds stimulate adenosine diphosphate (ADP)-ribosylation of a 41 kDa cytosolic protein and regulate the glyceraldehyde-3-phospate dehydrogenase activity. Yet, although D. discoideum cells produce NO at a relatively constant rate at the onset of their developmental cycle, there is still no evidence of the presence of nitric oxide synthase (NOS) enzymes. In this work, we detect the nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity in D. discoideum and we characterise it by specific inhibitors and physical-chemical conditions that allegedly distinguish between NOS-related and -unrelated NADPH-d activity.Key words: NADPH-diaphorase activity, protozoa, nitric oxide synthase.     

Inhibition of cell differentiation and cell cohesion by tunicamycin in Dictyostelium discoideum

The effects of tunicamycin on protein glycosylation and cell differentiation were examined during early development of Dictyostelium discoideum. Tunicamycin inhibited cell growth reversibly in liquid medium. At a concentration of 3 μg/ml, tunicamycin completely inhibited morphogenesis and cell differentiation in developing cells. These cells remained as a smooth lawn and failed to undergo chemotactic migration. The expression of EDTA-resistant contact sites was also inhibited. The inhibition by tunicamycin was reversible if cells were washed free of the drug within the first 10 hr of incubation. After 12 hr of development, cells were protected from the drug by the sheath. When cells were treated with tunicamycin during the first 10 hr of development, incorporation of [³H]mannose and [³H] fucose was inhibited by approximately 75% within 45 min while no significant inhibition of [³H]leucine incorporation was observed during the initial 3 hr of drug treatment. The inhibition of protein glycosylation was further evidenced by the reduction in number of glycoproteins “stained” with ¹²⁵I-labelled con A. A number of developmentally regulated high-molecular-weight glycoproteins, including the contact site A glycoprotein (gp80), were undetectable when cells were labelled with [³H]fucose in the presence of tunicamycin. It is therefore evident that glycoproteins with N-glycosidically linked carbohydrate moieties may play a crucial role in intercellular cohesiveness and early development of D. discoideum.     

Cyclic AMP Phosphodiesterase and its Inhibitor in Slime Mould Development

CYCLIC adenosine-3′,5′-monophosphate (cyclic AMP) acts as a chemotactic factor causing cell aggregation in the slime mould, Dtctyosteltum discoideum^1,2. Aggregation in this organism is the link between the growth phase and the second phase of development, in which cells cooperate and differentiate to form a multicellular fruiting body. The finding that cyclic AMP also mediates developmental functions other than chemotaxis³ suggests that regulation of cyclic AMP synthesis and destruction is important in the control of morphogenesis in D. discoideum.     

Differentiation and dedifferentiation can function simultaneously and independently in the same cells in Dictyostelium discoideum

When developing cultures of Dictyostelium discoideum are disaggregated and morphogenesis is reinitiated, cells recapitulate the stages they had progressed through prior to disaggregation in a fraction of the original time. If developing cultures are disaggregated and the cells resuspended in nutrient medium, they retain this capacity for 1.5 hr and then synchronously and rapidly revert to the slow timing of log phase cells. Loss of the capacity to recapitulate morphogenesis rapidly is referred to as the “erasure event.” Following the erasure event, cells systematically lose developmentally acquired functions in a defined temporal sequence of dedifferentiation. Cells which have just passed through the erasure event can be stimulated to reenter the developmental program, even though they still possess several aggregation-associated functions acquired during the initial developmental program. In this report, we have tested whether cells stimulated to reenter the developmental program immediately after the erasure event progress along the same rate-limiting pathway leading to aggregation as they did during initial development and whether this rate-limiting pathway can run simultaneously with and independently of the sequence of dedifferentiation. Results are presented which demonstrate (1) that the erasure event resets the rate-limiting pathway for development back to zero and that erased cells reentering development progress along the same rate-limiting pathway as naive log phase cells, (2) that the loss of an aggregation-associated function late in the sequence of dedifferentiation is completely blocked by the addition of cycloheximide, but not cAMP, just prior to the expected time of loss, and (3) that differentiation and dedifferentiation can function simultaneously and independently in the same cells, even though the former leads to the acquisition and the latter to the loss of the same aggregation-associated functions (in this case EDTA-resistant adhesion and cAMP-stimulated motility).     

Monoclonal antibody characterisation of slime sheath: the extracellular matrix of Dictyostelium discoideum

Proteins can be extracted from the slime sheath of Dictyostelium discoideum slugs by denaturing agents. A subset of these proteins is also released by cellulase digestion of the sheath, implying that protein-protein and protein-cellulose interactions are involved in sheath protein retention. It seems probable that the cellulose-associated sheath proteins are also associated with the cellulose of mature stalk cells. Monoclonal antibodies directed against sheath demonstrate extensive sharing of antigenic determinants between sheath proteins and a limited degree of antigenic sharing between sheath and slug cell proteins. All of the proteins recognised by these monoclonal antibodies are developmentally regulated. These results are discussed in terms of the structure of the sheath and its possible role(s) in D. discoideum development.     

The role of the plasma membrane in the development of Dictyostelium discoideum. II. Developmental and topographic analysis of polypeptide and glycoprotein composition

Previous workers have shown in a variety of ways that cell contact is required for the differentiation of Dictyostelium discoideum. Because interactions between cells are probably mediated by molecules on their plasma membranes, we have characterized the polypeptide composition of the membrane of cells at different stages of development. At least 55 polypeptides are found in the plasma membrane of vegetative cells. The polypeptide composition of the plasma membranes changes considerably during development. Treatment of intact cells with pronase indicated that many of the altered components appear to be located on the external surface of the plasma membrane where they could participate in interactions between cells. Similar digestion of the isolated membranes destroys most of their polypeptides, indicating that the bulk of the proteins of the plasma membrane are not completely embedded in the membrane. Several polypeptides appear to change in sensitivity to pronase during development. There are several changes in glycoprotein composition which occur between log phase and aggregation phase. An almost complete change in glycoprotein species occurs between aggregation and pre-culmination. Unlike the polypeptides, the glycoproteins are very resistant to pronase treatment in intact cells. However, some are pronase sensitive in isolated membranes.