Expression of growth factors in Dictyostelium discoideum

Growth factors and their binding proteins are important proteins regulating mammalian cell proliferation and differentiation so there is considerable interest in producing them as recombinant proteins, especially in hosts that do not already produce a complex mixture of growth factors. Many growth factors require post-translational modifications making them unsuitable for production in Escherichia coli or other prokaryotes. Since several expression vector systems have been recently developed for foreign protein production in the cellular slime mould, Dictyostelium discoideum, we attempted to use two of these systems to express human insulin-like growth factor binding protein 6 (hIGFBP6) and bovine beta-cellulin (bBTC) as secreted proteins. Although both proteins were successfully produced in stably transformed amoebae, no secretion was detected in spite of several attempts to facilitate this occurring.     

Products of endogenous N-acetylglucosaminyltransferase activity of Dictyostelium discoideum during growth and early development.

In the presence of Mn2+ and uridine diphosphate-N-acetyl-D-[14C]glucosamine, a total particulate fraction prepared from Dictyostelium discoideum amoebae catalyzed the transfer of N-acetyl[14C]glucosamine to endogenous membrane protein acceptors. No transfer to lipid acceptors was evident. The 14C products obtained from growth-phase and aggregation-competent amoebae were converted to glycopeptides by pronase digestion. The respective glycopeptides appeared identical in their chemical and chromatographic properties, suggesting that the same activity was functioning in both growing and differentiating cells. The results provided no evidence for developmental regulation of this activity in D. discoideum.     

Properties and developmental regulation of an alpha-d-galactosidase from Dictyostelium discoideum.

An alpha-D-galactosidase was detected in cells of the cellular slime mould, Dictyostelium discoideum, at all stages of development. Its specific activity was highest during early development (interphase), and this accumulation of enzyme appears to require protein synthesis de novo. Its subcellular distribution differs from that of other D. discoideum glycosidases, since most activity was recovered in the soluble fraction. No evidence was obtained for more than one isoenzymic form after subjection of extracts to electrophoresis and various chromatographic procedures. It is excreted from the cell during development, but no evidence was found for an extracellular function for the enzyme.     

Role of cAMP-dependent protein kinase during growth and early development of Dictyostelium discoideum

cAMP-dependent protein kinase (PKA) is an essential regulator of gene expression and cell differentiation during multicellular development of Dictyostelium discoideum. Here we show that PKA activity also regulates gene expression during the growth phase and at the transition from growth to development. Overexpression of PKA leads to overexpression of the discoidinIgamma promoter, while expression of the discoidinIgamma promoter is reduced when PKA activity is reduced, either by expression of a dominant negative mutant of the regulatory subunit or by disruption of the gene for the catalytic subunit (PKA-C). The discoidin phenotype of PKA-C null cells is cell autonomous. In particular, normal secretion of discoidin-inducing factors was demonstrated. In addition, PKA-C null cells are able to respond to media conditioned by PSF and CMF. We conclude that PKA is a major activator of discoidin expression. However, it is not required for production or transduction of the inducing extracellular signals. Therefore, PKA-dependent and PKA-independent pathways regulate the expression of the discoidin genes.     

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.     

Expression of glycosidase activities during germination of Dictyostelium discoideum spores.

Several lysosomal glycosidase activities were examined in vitro during heat-induced germination of Dictyostelium discoideum spores and were found not to be coordinately controlled. The level of beta-glucosidase activity increased significantly during the emergence stage of germination. Both alpha-glucosidase and N-acetyl-beta-glucosaminidase activities remained relatively constant until postemergence, when they increased slightly; alpha-mannosidase activity decreased during all stages of germination. The activity of beta-galactosidase increased slightly during spore swelling, fell below the level initially found in spores at zero time, and increased slightly during postemergence. The expression of all of these enzyme activities, except the increase in beta-galactosidase, appeared to require protein synthesis. Spores in the lag phase of germination which were exposed to severe environmental stress were deactivated and exhibited reduced levels of alpha-glucosidase, beta-glucosidase, and N-acetyl-beta-glucosaminidase activities. Prolonged heat activation treatment reduced the levels of lysosomal glycosidase activities in postactivated spores but did not change the subsequent enzyme patterns during the spore-swelling and emergence stages of germination.     

Signal emission and signal propagation during early aggregation in Dictyostelium discoideum.

Waves of chemotactic movement during the early phase of aggregation in Dictyostelium discoideum are of 2 kinds, concentric waves produced by cells that emit cyclic AMP signals spontaneously, and spirals generated by excitations relayed continuously around loops of excitable cells. The period of a spiral wave is the time taken for the excitation to make one complete circuit of the pacemaker loop. We have compared signal emission from the 2 types of source in time-lapse films made at a variety of temperatures. Our results show that spiral waves have a characteristic period length throughout most if not all of the early phase of aggregation, and that the period of concentric waves is generally longer and more variable. Temperature has a pronounced effect on period length and a lesser effect on propagation velocity. We find that each individual wave is propagated at constant velocity over distances of 1-2 cm but that the velocity of successive waves declines. This decline probably reflects some cumulative effect of the chemotactic excitations on the excitable properties of the aggregating cells.     

Expression of proteolytic enzymes during Dictyostelium discoideum spore germination

The specific activity of cathepsin B-like, cathepsin D-like, and leucine aminopeptidase enzymes was measured in dormant, aging, and germinating spores of wild-type and mutant Dictyostelium discoideum.The activity of leucine aminopeptidase was relatively constant during spore aging and spore germination. The level of cathepsin D-like activity was highest in young dormant spores but decreased during germination or aging.The level of cathepsin B-like activity remained constant in wild-type spores which were aged for 13 days. The dormant spores of spontaneous germination mutants initially contained low levels of cathepsin B-like activity which increased during aging. Thus, there was no correlation between the level of endogenous cathepsin B activity and the ability to be autoactivated or heat-activated. The level of cathepsin B-like activity does not have a role in the generation of energy for the swelling stage of germination. Finally, the combined level of endogenous and exogenous cathepsin B activity increased more than 20-fold during the emergence of myxamoebae suggesting that the enzyme(s) may play a role at this development stage of germination.     

An analysis of developmental timing in Dictyostelium discoideum

A new method has been developed to assess the minimum complexity and relationships of those pathways (developmental timers) which time the consecutive stages of a developing system (Soll, 1983). This method has been applied to the morphogenetic program of Dictyostelium discoideum and has resulted in (1) a minimum estimate of the number of components comprising the timers for the first seven stages of morphogenesis, (2) a characterization of the temperature sensitivities of these components including demonstration of a reversible timer component, (3) detailed temporal definition of a number of transition points between rate-limiting components including a major branch point for the onset of several independent timer components coincident with the onset of aggregation, and (4) a temporal model for the relationships between the timers of the seven consecutive morphogenetic stages, including several examples of parallel timers.     

Correlates of developmental cell death in Dictyostelium discoideum

We have studied the correlates of cell death during stalk cell differentiation in Dictyostelium discoideum. Our main findings are four. (i) There is a gradual increase in the number of cells with exposed phosphatidyl serine residues, an indicator of membrane asymmetry loss and increased permeability. Only presumptive stalk cells show this change in membrane asymmetry. Cells also show an increase in cell membrane permeability under conditions of calcium-induced stalk cell differentiation in cell monolayers. (ii) There is a gradual fall in mitochondrial membrane potential during development, again restricted to the presumptive stalk cells. (iii) The fraction of cells showing caspase-3 activity increases as development proceeds and then declines in the terminally differentiated fruiting body. (iv) There is no internucleosomal cleavage of DNA, or DNA fragmentation, in D. discoideum nor is there any calcium- and magnesium-dependent endonucleolytic activity in nuclear extracts from various developmental stages. However, nuclear condensation and peripheralization does occur in stalk cells. Thus, cell death in D. discoideum shows some, but not all, features of apoptotic cell death as recognized in other multicellular systems. These findings argue against the emergence of a single mechanism of 'programmed cell death (PCD)' before multicellularity arose during evolution.     

Developmentally regulated protein-tyrosine kinase genes in Dictyostelium discoideum.

Dictyostelium discoideum, an organism that undergoes development and that is amenable to biochemical and molecular genetic approaches, is an attractive model organism with which to study the role of tyrosine phosphorylation in cell-cell communication. We report the presence of protein-tyrosine kinase genes in D. discoideum. Screening of a Dictyostelium cDNA expression library with an anti-phosphotyrosine antibody identifies fusion proteins that exhibit protein-tyrosine kinase activity. Two distinct cDNAs were identified and isolated. Though highly homologous to protein kinases in general, these kinases do not exhibit many of the hallmarks of protein-tyrosine kinases of higher eucaryotes. In addition, these genes are developmentally regulated, which suggests a role for tyrosine phosphorylation in controlling Dictyostelium development.     

Temporal and spatial expression of ammonium transporter genes during growth and development of Dictyostelium discoideum

Ammonia is an important signaling molecule involved in the regulation of development in Dictyostelium. During aggregation, ammonia gradients are established, and the ammonia concentration in the immediate environment or within a particular cell throughout development may vary. This is due to the rate of cellular ammonia production, its rate of loss by evaporation to the atmosphere or by diffusion into the substratum, and perhaps to cellular transport by ammonium transporters (AMTs). Recent efforts in genome and cDNA sequencing have identified three ammonium transporters in Dictyostelium. In addition to physically altering the levels of ammonia within cells, AMTs also may play a role in ammonia signaling. As an initial step in identifying such a function, the temporal and spatial expression of the three amt genes is examined. RT-PCR demonstrates that each of the three amt mRNAs is present and relatively constant throughout growth and development. The spatial expression of these three amt genes is examined during multiple stages of Dictyostelium development using in situ hybridization. A distinct and dynamic pattern of expression is seen for the three genes. In general, amtA is expressed heavily in pre-stalk cells in a dynamic way, while amtB and amtC are expressed in pre-spore regions consistently throughout development. AmtC also is expressed in the most anterior tip of fingers and slugs, corresponding to cells that mediate ammonia's effect on the choice between slug migration and culmination. Indeed, amtC null cells have a slugger phenotype, suggesting AmtC functions in the signaling pathway underlying the mechanics of this choice.     

Effects of BUdR on developmental functions of Dictyostelium discoideum.

The development of Dictyostelium discoideum cells, as measured by spore yield, is somewhat more sensitive to the presence of BUdR during vegetative growth than is growth itself. Observations on the development of control and BUdR-grown cells, their protein labelling patterns and assays of 4 developmentally regulated proteins all reveal a consistent picture. BUdR appears to block spore formation by partially inhibiting several or many different earlier events during development. The relative sensitivity of development compared to growth to inhibition by the drug may be a consequence of the nature of the developmental process rather than of some unique specificity of the inhibitor.     

Characterization and developmental regulation of beta-galactosidase isozymes in Dictyostelium discoideum.

Two isozymes of β-galactosidase (EC 3.2.1.23) have been identified during growth and development of the cellular slime mold, Dictyostelium discoideum. The isozymes have been partially purified and differ in a variety of physical and enzymatic properties. β-Galactosidase-1 is present in vegetative cells. The specific activity is reduced during early development and then increases again during culmination. The specific activity of β-galactosidase-2 increases in early development and then again during culmination and spore maturation. The specific activity of β-galactosidase-2 is extremely dependent upon growth conditions and is regulated over a 160-fold range. The accumulation of both isozymes is dependent on concomitant RNA and protein synthesis.     

The effects of cell density and starvation on early developmental events in Dictyostelium discoideum

We show that removal of yeast extract and trypticase from growth medium is sufficient for induction of several key events which occur during the early stages of Dictyostelium differentiation: run-off of polysomes, the earliest known change in macromolecular metabolism; appearance of the cell surface cAMP receptor; and aggregation itself. Starvation of glucose has little effect on these parameters. These results are consistent with those of other investigators who showed that starvation only of amino acids will induce other activities associated with cAMP-mediated cell signaling and cell-cell adhesion. We show, in contrast, that other factors are involved in the increase in the relative rates of synthesis of three polypeptides very early in differentiation: actin, and two proteins (“45-min” proteins) which are synthesized only during the period of 45–90 min. The induction of synthesis of these three proteins and presumably, of their mRNAs, is not the result of starvation for glucose or amino acids but is the result of plating cells at high density. The increases in the synthesis of these proteins are dependent on the density at which cells are plated and do not occur at a density 75-fold lower than the density used in standard experiments. Cells growing at high density or near stationary phase do not show the induction of increased synthesis of actin or the “45-min” proteins. These experiments suggest that these early developmental changes may be dependent on a threshold level of a diffusible factor excreted early in development.