A prespore-cell-inducing factor in Dictyostelium discoideum: its purification and characterization.

Under starvation conditions, amoebae of Dictyostelium discoideum aggregate to form multicellular masses; the aggregates are then initiated to differentiate. We have reported previously that a signal substance exists in conditioned medium of D. discoideum, and we named it prespore-cell-inducing factor (psi, Psi factor) [Oohata, Nakagawa, Tasaka, and Fujii (1997) Development 124, 2781-2787]. The factor can induce isolated amoebae to differentiate into prespore cells. Moreover, we suggested that it caused not only cell differentiation but also cell division. In the present study, we have purified Psi factor from the conditioned medium and characterized it. The purified Psi factor induced both prespore cell differentiation and cell division of prespore cells. Its apparent molecular mass was 180 kDa by gel filtration and 106 kDa by SDS/PAGE. Based on these results, Psi factor exists as a dimer in normal conditions. Periodic acid/Schiff staining showed that Psi factor was a glycoprotein. It was ascertained by Edman degradation that Psi factor is blocked at the N-terminal. Treatment with pyroglutamate aminopeptidase removed the N-terminal block and allowed determination of the amino-acid sequence of Psi factor. Moreover, three internal amino-acid sequences were determined in limited proteolysis experiments using trypsin and endoproteinase Lys-C. The homology search for these sequences supports the fact that Psi factor is a novel differentiation factor.

A novel prespore-cell-inducing factor in Dictyostelium discoideum induces cell division of prespore cells.

In Dictyostelium discoideum strain V12M2, at a very low cell density (approximately 10(2) cells/cm2), most amoebae differentiate into prespore cells in a salt solution containing cAMP if an adequately diluted conditioned medium (CM) is provided (Oohata, A. A. (1995) Differentiation 59, 283-288). This finding suggests the presence of factor(s) released into the medium that are involved in inducing prespore cell differentiation. In the present study, we report the presence of two types of factors that function synergistically in prespore cell induction; one is a heat-stable and dialysable factor(s) and the other is a heat-labile and non-dialysable factor termed psi (psi) factor (prespore-inducing factor). We purified and characterized the psi factor. Its relative molecular mass was determined to be 106x10(3) Mr by SDS-PAGE and 180x10(3) Mr by gel filtration HPLC, respectively. These results indicate that psi factor exists as a dimer under native conditions. In addition to inducing prespore cell differentiation, psi factor induced cell division of prespore cells in submerged culture. Our results suggest that psi factor plays important roles not only in prespore cell differentiation but also in the progress of the cell cycle in the prespore pathway in normal development.

Factors controlling prespore cell differentiation in Dictyostelium discoideum: minute amounts of differentiation-inducing factor promote prespore cell differentiation.

Amoebae of strain V12M2 differentiate efficiently into prespore cells without cell contact in a salt solution containing cAMP, if the pH of the medium is maintained suitably acidic using a restricted buffer [31]. Under such conditions, most cells differentiate into prespore cells at pH 5.2. Using this developmental system, the elements controlling prespore-cell differentiation were analyzed. First, the dependence on cell density was examined. At a very low density (10(2) cells/cm2), most cells did not differentiate. As the density was increased the proportion of prespore cells differentiating increased, reaching a maximum at 5 x 10(3) cells/cm2. Conditioned medium could mimic the effects of cell density on cell differentiation. These findings suggest the presence of factor(s) released into the medium which are involved in inducing prespore-cell differentiation. The conditioned medium was found to contain at least two prespore-inducing components; one is a novel factor(s) and the other is DIF, which has previous only been considered to repress prespore-cell differentiation. These findings were supported by experiments using a DIF-deficient mutant, HM44.

Role of cyclic AMP and ammonia in induction and maintenance of post-aggregative differentiation in a suspension culture of Dictyostelium discoideum.

The effects of ammonia and cAMP on prespore and prestalk differentiation of Dictyostelium discoideum were investigated by monitoring eight developmentally regulated proteins as differentiation markers under the shake culture conditions in glucose/albumin medium. In the medium containing cAMP, cells form small agglomerates and undergo prespore differentiation [19]. Under the conditions where agglomeration was prevented, ammonia induced four marker proteins out of eight tested in the presence of cAMP, which included not only a prespore specific enzyme but also cell-type non-specific proteins. No inhibitory effect of ammonia was observed in presumptive cell differentiation. These results suggest that ammonia is an inducer of differentiation at the protein level as well as the mRNA level as found previously [24]. The effects of cAMP were examined with special attention to the difference between induction of differentiation and maintenance of differentiated state in this specific medium. The induction of differentiation from early aggregative cells was cAMP-dependent with all the marker proteins tested. This agrees with the observations so far obtained in other culture systems. However, when already differentiated cell masses (slugs) were dissociated and shaken in this specific medium, only two enzymes required cAMP to maintain the activity while five out of eight kinds of the proteins continued to be expressed as in undisturbed slugs even without cAMP. This suggests that for the maintenance of the differentiated state after slug disaggregation cAMP may not be required with respect to the majority of proteins, if cells are provided with some favorable conditions such as glucose/albumin medium.

Multiple forms of acid phosphatase and their differential secretion during stalk formation under submerged monolayer incubation of Dictyostelium discoideum.

The electrophoretic pattern of intracellular and secreted acid phosphatases (AcPases) in Dictyostelium discoideum was examined during incubation of the cells as a submerged monolayer. Three distinct forms of the enzyme were observed in the cell during differentiation; one was detected throughout development (AcPase 1), whereas the others including AcPase 2 were stage-specific. AcPase 1 was released in the medium predominantly in early development and AcPase 2, a prestalk specific form, was secreted during stalk formation. When cells were incubated under conditions where stalk cells did not form, only AcPase 1 was recognized both in the cell and in the medium.

Dictyostelium mutants lacking DIF, a putative morphogen.

DIF is an endogenous extracellular signal that may control differentiation of D. discoideum cells. It is a dialyzable, lipid-like factor that induces stalk cell formation among isolated amebae incubated in vitro with cAMP. To examine the consequences of DIF deprivation, we have isolated several mutant strains that are impaired in DIF accumulation, and whose inability to make stalk cells in vitro and during normal development on agar can be corrected by the addition of exogenous DIF. Little DIF is made by the mutants, and morphological development on agar stops after the cells have aggregated, but before a slug forms. In these DIF-deprived conditions, prespore cells can differentiate, but prestalk cells cannot.

A prestalk-cell-specific acid phosphatase in Dictyostelium discoideum.

A simple and reliable method of separating and quantifying acid phosphatase activities in D. discoideum is described, and one of the electrophoretically distinguishable forms of the enzyme is shown to be specific for prestalk cells. Accumulation of this product commences some 2-3 h later in development than the prespore specific enzyme UDP-galactose polysaccharide transferase. This finding is discussed in terms of recent observation on the control of prestalk cell differentiation in this organism.

Separation and biochemical characterization of the two cell types present in the pseudoplasmodium of Dictyostelium mucoroides.

The pseudoplasmodium (slug) of the cellular slime mould, Dictyostelium mucoroides consists of prestalk and prespore cells. These 2 differentiated types of cells were separated by modification of the previous methods using density-gradient centrifugation. Major improvements made in the present study were the use of a density column of different specific gravities and the use of a discontinuous gradient rather than a continuous one. With these improvements, it became possible to obtain efficiently a large number of prestalk and prespore cells. After separation of the 2 types of cells, activities and electrophoretic patterns of some developmentally regulated enzymes were compared. The hydrolases such as beta-glucosidase, beta-galactosidase, acetylglucosaminidase and alkaline phosphatase showed higher activities in the prestalk than in the prespore cells. The results are consistent with the fact that more autophagic vacuoles are present in the prestalk than in the prespore cells. On the other hand, UDP-galactose polysaccharide transferase was almost exclusively found in the prespore cells. Electrophoresis on polyacrylamide gels of slug, prestalk and prespore extracts showed that one among 4 isozymes of beta-galactosidase recognized in the slug extract was present only in the prestalk extract. Electrophoretic patterns of acid phosphatase revealed that one of the two isozymes present in the slug was specifically found in the prestalk cell. Finding of such prestalk specific isozymes was significant, since no specific markers have been known for the prestalk cell.