Changes of plasma membrane proteins during prespore differentiation in Dictyostelium discoideum

By the use of a shake culture system, we have previously shown (Oyama, M., Okamoto, K., & Takeuchi, I. (1982) J. Cell Sci. 56, 223-232) that both cAMP and cAMP-dependent cell contact are required for prespore differentiation in Dictyostelium discoideum. The present study was undertaken to examine changes of the plasma membrane proteins during prespore differentiation in the shake culture system. Rabbit antibodies prepared against the plasma membrane fraction of the differentiated cells inhibited the reaggregation of the differentiated cells but not that of aggregation-competent cells. This result indicates that new contact sites are formed in the differentiated cells. By the combined use of the antibody-conjugated immuno-adsorbent with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, changes of membrane proteins were analyzed with the cells incubated under various conditions. Three proteins were found to be present specifically in the differentiated cells only in the presence of cAMP, one of which (105K protein) appeared when cells became adhesive, but before prespore specific proteins were detected. Two others (80K and 58K proteins) appeared during prespore differentiation after cells formed agglomerates.     

Unexpected localisation of cells expressing a prespore marker of Dictyostelium discoideum

Abstract. We show that the anterior, prestalk region of the Dictyostelium slug contains cells which express, or have expressed, a prespore-specific marker. We term these cells "prespore-like cells" (PLC). In newly formed slugs there is a sharp prespore/prestalk boundary, with very few PLC, but after several days of migration the clear demarcation between prespore and prestalk zones breaks down because the number of PLC increases dramatically. This is consistent with previous observations showing there to be rapid interchange of cells between the prestalk and prespore regions. This is not, however, their only source, as a scattering of PLC appear when separate prestalk and prespore regions first become apparent at the time of tip formation. Also, at culmination, there is respecification of "prespore" cells at the pre-stalk/prespore boundary to form part of the mature stalk. The existence of these cells, and of PLC, may explain why we find prespore-specific mRNAs in mature stalk cells.     

Differential Cell Cohesiveness Expressed by Prespore and Prestalk Cells of Dictyostelium discoideum

Pseudoplasmodia of Dictyostelium discoideum at the culmination stage were separated into two cell populations by sedimentation in a discontinuous renografin gradient. The two lighter fractions (I and II) had enzymatic activities characteristic of the anterior prestalk cells, while the heaviest fraction (III) showed enzyme activities characteristic of the posterior prespore cells. Cell-cell adhesion among prespore cells is much more resistant to EDTA dissociation than 10-h cells and prestalk cells. Fab fragments prepared from antibodies directed against a specific cell surface glycoprotein gp150 were more effective in dissociating prespore cells than prestalk cells. In addition, prespore cells contained an approximately 2-fold higher concentration of the endogenous carbohydrate binding protein discoidin-I than prestalk cells. These differences may account for the differential cohesiveness of these two cell populations and provide a basis for cell recognition and cell sorting at the slug stage.     

Re-expression of 117 antigen, a cell surface glycoprotein of aggregating cells, during terminal differentiation of Dictyostelium discoideum prespore cells

117 antigen is a glycoprotein expressed on the surface of D. discoideum cells at aggregation. It then disappears and is later re-expressed on the surface of a subpopulation of cells at culmination, the terminal differentiation stage (Sadeghi et al. 1987). A cDNA clone was used to show that the appearance of cell surface 117 antigen accurately reflects the expression of the 117 gene as measured by mRNA levels. It was also shown that during multicellular development there is a reciprocal relationship between the levels of 117 mRNA and the mRNA which codes for prespore surface glycoprotein, PsA. Dual parameter flow cytometry was used to demonstrate that the 117 antigen is found on the surface of maturing prespore cells after the PsA glycoprotein disappears, but that it is not found on mature spores. Using three monoclonal antibodies which identify respectively 117 antigen, PsA, and MUD3 antigen (a spore coat glycoprotein--probably Sp96), two new stages of final spore maturation were defined. These results indicate that there is a recapitulation of at least one aggregative cell surface glycoprotein in the prespore subpopulation of cells as they rise up the stalk during final spore development. This raises the possibility that culmination, which involves complex three dimensional morphogenetic movements not unlike those observed during animal embryogenesis, involves components of the two-dimensional pattern seen during aggregation.     

A stalk-specific localization of trehalase activity in Dictyostelium discoideum.

By utilizing ultra-microtechniques, trehalase activity was followed in specific cell types during the differentiation cycle of Dictyostelium discoideum. When whole organisms were assayed, trehalase activity was found to be high in the early stages of differentiation, decreased to its lowest point at 14 h, and then increased at the end of the cycle. By microdissection of freeze-dried individuals, the activity of trehalase could be followed during the migration of pre-stalk and pre-spore cells. No activity was observed at any stage of spore cell development, whereas stalk cells showed a rapid increase in activity upon maturation. An increasing gradient of activity was found from the apex of the stalk toward the base. This localization of trehalase in stalk cells resolves some contradictory results in the literature concerning the role of the enzyme during differentiation.     

Prestalk/prespore Differentiation of Dictyostelium Cells under the Conditions Favoring Stalk or Spore Cell Formation*

The differentiation processes of Dictyostelium discoideum cells under the conditions which favored either stalk or spore cell formation were examined by the use of prestalk- and prespore-specific antibodies. In stalk cell-forming conditions, cells reactive with prestalk-specific monoclonal antibody (C1) increased rapidly early in development and later differentiated into stalk cells. No or only a few cells became reactive with prespore-specific monoclonal (B6) and polyclonal (antispore) antibodies. Despite the fact that most cells terminally became spores under spore cell-forming conditions, cells were first stained with the C1 antibody before becoming reactive with the B6 antibody. Unlike the case of normal development where cells coincidentally become reactive with the B6 and antispore antibodies, the appearance of the cells reactive with the latter was either delayed or suppressed. In conclusion, under either spore or stalk cell-forming conditions, the appearance of the prestalk antigen preceded that of the prespore one, which is consistent with normal development.     

Production and turnover of cAMP signals by prestalk and prespore cells in Dictyostelium discoideum cell aggregates

Dictyostelium discoideum prestalk cells and prespore cells from migrating slugs and culminating cell aggregates were isolated by Percoll density centrifugation. Several activities relevant to the generation, detection, and turnover of extracellular cyclic AMP (cAMP) signals were determined. It was found that: the two cell types have the same basal adenylate cyclase activity; prespore cells and prestalk cells are able to relay the extracellular cAMP signal equally well; intact prestalk cells show a threefold higher cAMP phosphodiesterase activity on the cell surface than prespore cells, whereas their cytosolic activity is the same; intact prestalk cells bind three to four times more cAMP than prespore cells; no large differences in cAMP metabolism and detection were observed between cells derived from migrating slugs and culminating aggregates. The results are discussed in relation to the possible morphogenetic role of extracellular cAMP in Dictyostelium cell aggregates. On the basis of the properties of the isolated cells we assume that a gradient of extracellular cAMP exists in Dictyostelium aggregates. This gradient appears to be involved in the formation and stabilization of the prestalk-prespore cell pattern.     

Spore coat proteins of Dictyostelium discoideum are packaged in prespore vesicles

Previous studies have shown that Dictyostelium discoideum spore coat proteins are found in prespore cells, which are localized to the posterior region of migrating slugs, and in the coats of mature spores. Prespore vesicles, identified by morphology and by staining with anti-D. mucoroides spore serum, are also localized in the posterior region of migrating slugs. Using antisera specific to the spore coat proteins, we show that the spore coat proteins are packaged in prespore vesicles. They are present in the vesicles as a complex which can be dissociated by denaturation. The anti-D. mucoroides spore serum reacts with at least five proteins in whole spore extracts including the spore coat proteins SP96 and SP70.     

Cytokeratin filament expression during in vitro teratocarcinoma cell differentiation as detected by a monoclonal antibody

Undifferentiated F9 teratocarcinoma cells were induced to differentiate in culture using retinoic acid and cAMP. As a result the morphology of the cultures changes dramatically. Using a monoclonal antibody directed against cytokeratin polypeptide 18 (RGF 53) in the indirect immunofluorescence technique we could show that this cytokeratin subunit is synthesized and assembled into a filamentous network upon differentiation in about 50% of the cells. Immunoblotting studies confirm these results.     

Cytokeratin filament expression during in vitro teratocarcinoma cell differentiation as detected by a monoclonal antibody

Undifferentiated F9 teratocarcinoma cells were induced to differentiate in culture using retinoic acid and cAMP. As a result, the morphology of the cultures changes dramatically. Using a monoclonal antibody directed against cytokeratin polypeptide 18 (RGE 53) in the indirect immunofluorescence technique we could show that this cytokeratin subunit is synthesized and assembled into a filamentous network upon differentiation in about 50% of the cells. Immunoblotting studies confirm these results.     

Calcium stores in differentiated Dictyostelium discoideum: prespore cells sequester calcium more efficiently than prestalk cells

Dictyostelium discoideum pseudoplasmodia exhibit a gradient of the cytosolic free Ca2+-concentration ([Ca2+]i) along their anterior-posterior axis involved in cell-type specific differentiation. [Ca2+]i is high in prestalk and low in prespore cells. We determined the content and localization of calcium and other elements in cryosectioned cells of pseudoplasmodia and fruiting bodies by X-ray microanalysis. Granular stores rich in Ca, Mg and P were identified. Average Ca was higher in prespore than prestalk granules (225vs 111 mmol/kg dry weight). Total Ca stored in granules was also higher in prespore than prestalk cells. The amount of P and S in granules differed between the two cell types indicating different store composition. In spores mean granular Ca was 120 mmol/kg dry weight. Stalk cells had smaller granules with 360 mmol Ca/kg dry weight. Complementary to microanalysis, vesicular Ca2+-fluxes were studied in fractionated cell homogenates. The rate of Ca2+-uptake was higher in pellet fractions of prespore than prestalk amoebae (4.7 vs 3.4 nmol/min x mg). Ca2+-release was greater in supernatant fractions from prestalk than prespore cells (16.5vs 7.7 nmol/10(8)cells). In summary, prestalk and prespore cells possess qualitatively different, high-capacity stores containing distinct amounts of Ca and probably being involved in regulation of the anterior-posterior [Ca2+]i-gradient.     

Cell behavior during formation of prestalk/prespore pattern in submerged agglomerates of Dictyostelium discoideum

When cells dissociated from Dictyostelium discoideum slugs were cultured in roller tubes, they formed agglomerates in which prestalk cells were initially dispersed but soon sorted out to the center and then moved to the edge to reconstitute the prestalk/prespore pattern. To examine the mechanism of sorting out, individual prestalk cells were traced by a videotape recorder. The radial component of the rate of movement toward the center of the presumptive prestalk region was calculated. Prestalk cells did not move randomly, but rather directionally toward the center. Their movement was pulsatile, with a period of ca. 15 min, and accompanied by occasional formation of cell streams, thus resembling the movement observable during cell aggregation. These results favor the idea that prestalk cells sort out to the prestalk region due to differential chemotaxis rather than differential adhesiveness. After formation of the prestalk/prespore pattern, the prestalk region rotated along the circumference of the agglomerates. This appears comparable to migration of slugs on the substratum, the rate of rotation being similar to that of slug migration. To examine the processes of pattern formation during development, washed vegetative cells were cultured in roller tubes. Prespore cells identified by antispore immunoglobulin initially appeared randomly within the agglomerates, but then nonprespore cells accumulated in the center and finally moved to the edge to establish the prestalk/prespore pattern, the processes being similar to those of pattern reconstruction with differentiated prestalk and prespore cells.     

Cellular and subcellular distribution of a cAMP-regulated prestalk protein and prespore protein in Dictyostelium discoideum: a study on the ontogeny of prestalk and prespore cells

We have analyzed a developmentally and spatially regulated prestalk-specific gene and a prespore-specific gene from Dictyostelium. The prestalk gene, pst-cathepsin, encodes a protein highly homologous to the lysosomal cysteine proteinases cathepsin H and cathepsin B. The prespore gene encodes a protein with some homology to the anti-bacterial toxin crambin and has been designated beejin. Using the lambda gtll system, we have made polyclonal antibodies directed against a portion of the protein encoded by pst-cathepsin and other antibodies directed against the beejin protein. Both antibodies stain single bands on Western blots. By immunofluorescence and Western blots, pst-cathepsin is not present in vegetative cells or developing cells during the first approximately 10 h of development. It then appears with a punctate distribution in a subset of developing cells. Beejin is detected only after approximately 15 h of development, also in a subset of cells. Pst-cathepsin is distributed in the anterior approximately 1/10 of migrating slugs and on the peripheral posterior surfaces of slugs. Beejin is distributed in the posterior region of slugs. Expression of both pst-cathepsin and beejin can be induced in subsets of isolated cultured cells by a combination of conditioned medium and extracellular cAMP in agreement with the regulation of the mRNAs encoding these proteins. We have used the antibodies as markers for cell type to examine the ontogeny and the spatial distribution of prestalk and prespore cells throughout multicellular development. Our findings suggest that prestalk cell differentiation is independent of position within the aggregate and that the spatial localization of prestalk cells within the multicellular aggregate arises from sorting of the prestalk cells after their induction. We have also found a class of cell in developing aggregates that contains neither the prestalk nor the prespore markers.     

A stalk-specific wheat germ agglutinin binding protein, wst34, in Dictyostelium discoideum can be detected with antiserum raised against Dictyostelium mucoroides stalk

A new stalk-specific wheat germ agglutinin (WGA) binding protein, wst34, has been identified in Dictyostelium discoideum and purified by the use of preparative sodium dodecyl sulfate - polyacrylamide gel electrophoresis and a WGA-affinity column. In normal development, wst34 appears during culmination and is maintained in stalk cells. It has a molecular mass of 34 kilodaltons and a pI value of 5.5-6.5. A polyclonal antiserum raised against stalk cell proteins of Dictyostelium mucoroides recognizes wst34 in western blots of D. discoideum proteins.     

Analysis of proportion regulation in slugs of Dictyostelium discoideum using a monoclonal antibody and a FACS-IV

A quantitative assay for estimating the proportion of prespore cells in D. discoideum slugs was established by labelling disaggregated slug cells with a prespore specific monoclonal antibody and analysing the cell population with a FACS-IV. The method is validated using a wild-type strain and its stalky mutant. "Wild-type" strains have different proportions of prespore cells and it is demonstrated that slugs of some strains have an increased percentage of prespore cells when migrated in the dark compared to the light and in the presence of EGTA. The technique is rapid and will make possible genetic analysis of proportion regulation in D. discoideum.     

A prespore gene, Dd31, expressed during culmination of Dictyostelium discoideum

During culmination of Dictyostelium fruiting bodies, prespore and prestalk cells undergo terminal differentiation to form spores and a cellular stalk. A genomic fragment was isolated by random cloning that hybridizes to a 1.4-kb mRNA present during culmination. Cell type separations at culmination showed that the mRNA is present in prespore cells and spores, but not in prestalk or stalk cells. After genomic mapping, an additional 3 kb of DNA surrounding the original 1-kb fragment was cloned. The gene was sequenced and named Dd31 after the size of the predicted protein product in kilodaltons. Accumulation of Dd31 mRNA occurs immediately prior to sporulation. Addition of 20 mM 8-Br-cAMP to cells dissociated from Mexican hat stage culminants induced sporulation and the accumulation of Dd31 mRNA, while 20 mM cAMP did not. Dd31 mRNA does not accumulate in the homeotic mutant stalky in which prespore cells are converted to stalk cells rather than spores. Characterization of Dd31 extends the known temporal dependent sequence of molecular differentiations to sporulation.