Origins of the prestalk-prespore pattern in Dictyostelium development.

Using cell-autonomous markers we have traced the origins of prespore cells and two types of prestalk cells (pstA and pstB cells) during slug formation. We show that cell sorting and positional information both contribute to Dictyostelium morphogenesis. The initial pattern established at the mound stage is topologically quite different from that of the slug. Confirming previous studies, we find that prespore cells occupy most of the aggregate but are absent from a thin layer at the base and from the emerging tip. PstB cells are almost entirely localized to the basal region during the early stages of tip formation. Thus prespore and pstB cell differentiation appear to occur in response to localized morphogenetic signals. In the case of pstB cells, these signals presumably emanate from the base and not, as might be expected, from the tip. When first detectable, pstA cells are scattered throughout the aggregate. They then appear to migrate to the apex, where the tip forms.

Two DIF-inducible, prestalk-specific mRNAs of Dictyostelium encode extracellular matrix proteins of the slug.

The migratory slug of Dictyostelium discoideum is surrounded by, and continuously synthesizes, an extracellular protein-cellulose matrix known as the slime sheath which is deposited on the substratum as a trail marking the slug's progress. We show that the stalk-specific proteins, ST310 and ST430, are exclusively located in the slime sheath and trail and that fusion genes, containing upstream sequences from the cognate genes, direct correct mRNA accumulation during development and correct localization of the fusion protein. Immunoelectron microscopy shows the ST310 and ST430 proteins to be present throughout the entire thickness of the slime sheath and almost totally absent from the cells of the slug. The genes that encode the ST310 and ST430 polypeptides are inducible by DIF, a stalk-specific inducing agent, and the mRNAs are highly enriched in prestalk over prespore cells. The production of these extracellular proteins by prestalk cells suggests that, in a manner somewhat analogous to that of extracellular matrix proteins of higher eukaryotes, the anterior region of the slug may be responsible for the continuous deposition of a track, along which the slug cells migrate. In the mature culminant, the ST310, and possibly the ST430, protein form part of the stalk tube and stalk cell wall. Therefore, the results also show that there are proteins common to both slime trial and stalk tube, indicating a possible precursor-product relationship between these chemically similar integuments.

Identification and localization of proteins encoded by two DIF-inducible genes of Dictyostelium.

We show that pDd56 and pDd63, two related DIF-inducible genes of Dictyostelium, respectively encode the ST310 and ST430 polypeptides identified by Morrissey, Devine, and Loomis (1984, Dev. Biol. 103, 414-424). We localize the two proteins by immunoelectron microscopy to the extracellular matrix surrounding the stalk cells and the stalk tube. Coupled with their predicted amino acid sequence and biochemical properties, this suggests that they are structural proteins of the stalk.

Structural and functional characterization of genes encoding Dictyostelium prestalk and prespore cell-specific proteins.

The nucleotide sequence of D19, a Dictyostelium gene that encodes a prespore-specific mRNA sequence shows it to encode PsA, the cell surface protein detected by the MUD 1 monoclonal antibody. The predicted sequence of the protein reveals a largely hydrophobic C terminus, with chemical similarity to proteins known to be attached to the plasma membrane via a phosphatidylinositol link. The C-terminal region has direct sequence homology to the contact sites A protein and to the phosphatidylinositol-linked form of a chicken N-CAM, suggesting that it might play a role in cell adhesion. Expression of the D19 gene is known to be induced by cAMP and repressed by adenosine. The accumulation of the D19 mRNA is also repressed by DIF, the putative stalk-specific morphogen, and this effect is mediated at the transcriptional level. The pDd56 and pDd63 genes are induced by DIF, and they are specific markers of prestalk and stalk cells. They encode, respectively, ST310 and ST430, two proteins that were first identified by two-dimensional gel electrophoresis. Both proteins are predominantly composed of a highly conserved, 24-amino acid repeat. The two proteins are localized in the slime sheath of the migratory slug and in the stalk tube and stalk cell wall of the mature culminant, where they presumably function as structural components of the extracellular matrix. We have constructed marked derivatives of the pDd56, pDd63, and D19 genes, and these are correctly regulated after transformation into Dictyostelium cells. Thus we have determined the structure, and elucidated possible functions, for one prespore and two prestalk genes. These sequences should be of value, both as markers of the earliest events in cellular differentiation and in identifying the regulatory sequences controlling cell type-specific gene expression.

Structural and functional characterization of a Dictyostelium gene encoding a DIF inducible, prestalk-enriched mRNA sequence.

The pDd56 mRNA sequence is highly enriched in prestalk over prespore cells and is inducible by DIF, the putative Dictyostelium stalk-specific morphogen. We show that the pDd56 gene is composed of forty one copies of a twenty four amino acid, cysteine rich repeat. This is highly homologus to a repeat which we have previously shown to compose the major fraction of the pDd63 mRNA, another DIF inducible, prestalk-enriched sequence. The predicted pDd56 protein contains a putative signal peptide but does not appear to contain a transmembrane segment. In combination these features suggest it to be an extrinsic protein and we confirm this elsewhere by showing that the pDd56 gene encodes a known, extracellular protein of the stalk. The pDd56 mRNA is dependent upon exogenous DIF for its accumulation. We show that this control is exerted at the transcriptional level and that a restriction fragment containing 1.7Kb of upstream sequence directs temporally-regulated expression of the gene.

Direct induction of Dictyostelium prestalk gene expression by DIF provides evidence that DIF is a morphogen.

We have isolated a gene that is very rapidly induced at the transcriptional level by DIF--a low molecular weight, diffusible factor necessary for stalk cell differentiation in Dictyostelium cells developing in vitro. The gene encodes a protein containing an N-terminal signal peptide preceding approximately 70 tandem repeats of a highly conserved 24 amino acid sequence with a high cysteine content. These features suggest it is an extracellular structural protein. During normal development, the gene is maximally expressed in the slug, in which the mRNA is very highly enriched in prestalk over prespore cells. The gene is not detectably expressed until the tipped aggregate stage, several hours later than prespore genes, suggesting that prespore cell differentiation precedes prestalk cell differentiation. The demonstration that DIF induces a gene normally only expressed in the prestalk zone of the slug provides strong evidence that DIF is a Dictyostelium morphogen.

Chemotaxis and cell motility in the cellular slime molds.

Chemotaxis and cell motility have essential roles to play throughout the developmental cycle of the cellular slime molds. The particular emphasis of this review, however, will be on the amoeboid stages of the life cycle. The nature of the chemoattractants and their detection will be discussed as will the possible mechanisms that may account for the directed locomotion of amoebae. Intracellular chemoattractant-elicited molecular responses thought to play a role in transduction of extracellular signals into a motility response will also be examined. Furthermore, relationships of these transduction pathway components with changes in assembly states of the cytoskeletal proteins contributing to shape change and cell movement will be assessed. Theories of amoeboid movement involving these cytoskeletal proteins will be compared and discussed in terms of their relevance to cellular slime mold motility.

TMB-8 inhibits respiration and cyclic GMP formation in Dictyostelium discoideum.

The putative inhibitor of intracellular calcium mobilization, TMB-8 was found to be a powerful inhibitor of respiration in amoebae of Dictyostelium discoideum. Consequently, the previously reported effects of this drug on cyclic GMP formation induced by chemoattractants were reassessed. It was found that TMB-8 abolished both folate and cyclic AMP-mediated accumulation of cyclic GMP in D. discoideum amoebae and that addition of Ca2+ completely restored this response. The Ca2+ chelating agent EGTA did not mimic the effect of TMB-8. The effect on cyclic GMP formation, however, occurred only at a concentration of TMB-8 that was ten times that causing maximal inhibition of respiration, and inhibition of cyclic GMP formation was completely restored by addition of excess Ca2+, whereas inhibition of respiration was only partially restored. The data suggest that TMB-8 has more than one inhibitory action, and because of the differential sensitivity of respiration and cyclic GMP formation to this drug, and the differential antagonism of excess Ca2+, we conclude that the effect of TMB-8 on the cyclic GMP response is probably due to its effect on Ca2+ mobilization, rather than indirectly via its effects on respiration. However, we advise caution in interpretation of data using this inhibitor where the responses measured are prolonged, are energy-requiring or are not freely reversible by excess Ca2+.

Effects of cytochalasin B on cell movements and chemoattractant-elicited actin changes of Dictyostelium.

The actin-binding drug cytochalasin B (CB) was employed to study the stability and role of cytoskeletal actin following chemotactic stimulation of Dictyostelium discoideum. Intact amoebae were found to be impermeable to this drug, as shown by lack of inhibition of chemotactic movement in its presence and failure of [3H]CB to bind to intact amoebae. However, there were approx. 150 000 high affinity CB-binding sites per cell detectable after cell breakage and preparation of Triton-insoluble cytoskeletons. The effect of CB on cytoskeletons was to destabilize the second (25-45 sec) and third (60 sec) chemotactically-induced peaks of cytoskeletal actin accumulation and to reduce the actin levels to the low prestimulus amount. In contrast, the drug had no such action on the rapid (3-5 sec) actin peak. This peak appeared to be stable in the presence of CB added before or simultaneously with lysis of the cell. It was also observed that the instability of the second and third peaks to CB gradually decreased after cell lysis (as did the number of CB binding sites) such that if CB was added 5 min after lysis of the chemotactically stimulated amoebae it had no destabilizing effect. Evidence was obtained from experiments employing centrifugation of cytoskeletons at 100 000 g and from the use of the DNase I inhibition assay for G-actin, that the first (3-5 sec) actin peak of accumulation involved polymerization rather than just cross-linking of short filamentous actin fragments. The significance of these actin accumulation peaks is discussed and their timing correlated with events involved in chemotaxis.

A new model for chemotactic signal transduction in Dictyostelium discoideum.

Dictyostelium discoideum amoebae were employed to study the refractoriness and adaptation of the rapid (5sec) accumulation of actin in their Triton-insoluble cytoskeletons following stimulation with specific chemoattractants. Amoebae became refractory within 10sec for this response but no adaptation occurred during this period. Amoebae desensitized for one attractant were not desensitized for another and responses to stimulation with a mixture of attractants were approximately additive. The characteristics of these processes are compared to published studies of adaptation in other chemoattractant-induced responses and a new model for the chemotactic signal transduction pathway is formulated. We conclude that intracellular cGMP accumulation may be on a separate branch of the pathway from the actin response.

Chemoattractant-mediated changes in cytoskeletal actin of cellular slime moulds.

We report changes in actin associated with the Triton-insoluble cytoskeleton following chemotactic stimulation in five species of cellular slime mould, Dictyostelium discoideum, D. mucoroides, D. purpureum, D. lacteum and Polysphondylium violaceum. Stimulation of amoebae with the chemoattractant specific for each species elicits a rapid increase in cytoskeletal actin content with a peak at 5 s, probably corresponding to pseudopodium formation. Subsequent changes consist of a second discrete peak of actin accumulation and, in at least two species, D. discoideum and P. violaceum, a third peak. We correlate these later changes in actin content with the changes in amoebal shape that have been reported to be a consequence of chemotactic stimulation. We have also investigated species variation in actin content and find D. purpureum to have a remarkably low cytoskeletal actin content in comparison to other species. Whole cell and cytoskeletal actin levels during the first 12 h of D. discoideum development have also been analysed. While little change is found in total protein content, whole cell actin shows an abrupt decline during aggregation and this is accompanied by an increase in cytoskeletal actin, amounting to 100% over 0-12 h of development. These results are discussed in the light of a possible changing role for actin during the course of development, from being involved in the dynamic events of chemotaxis to assuming a more structural role in the multicellular aggregate.

Changes in actin associated with the cytoskeleton following chemotactic stimulation of dictyostelium discoideum.

Chemotactic stimulation of Dictyostelium discoideum amoebae with pulses of cAMP or folate causes a series of rapid changes in the amount of actin protein associated with the Triton-insoluble cytoskeleton. The first of these changes occurs within 3 sec. of stimulation. The changes are dose-dependent and are within the physiological range of concentrations of cAMP or folate eliciting chemotaxis. These effects on the cytoskeleton show a pattern of regulation during development matching the respective chemotactic sensitivities of D.discoideum to cAMP (most sensitive at 4-8 hr of development) and to folate (rapidly decreasing sensitivity over 0-4 hr). At twelve hr, however, the responsiveness to folate unexpectedly reappears, suggesting a function of folate later in development than previously reported.