NC4 Cells Research Articles

A Maltose-binding-protein-like Peptide Accumulates after Mixing Two Types of Sexually Competent Cells of a Cellular Slime Mold, Dictyostelium discoideum.

Sexually competent cells of a cellular slime mold Dictyostelium discoideum strain NC4, were mixed with fusion-competent cells of either HM1, or WS2162, and mixed cell suspensions were incubated with rotation in the dark. Cells 30min after mixing NC4 and SW2162 contained giant cells and smaller cells. Mitochondria of giant cells were charastenstically distributed to the cell periphery. This trend was more evident in giant cells as nucleus number increased in a cell. At 24hr after mixing, blue fluorescences from cell wall, and dark brown fluorescence in the center of and around squashed structures were eminent, while both fluorescences were not detected in 30min cell. Western blot analysis using rabbit polyclonal antibody against bacterial maltose-binding protein detected a major 40 kD and a minor 43 kD peptides in total protein from cells at 24 and 66 hr after mixing NC4 and HN1 cells, while these bands were not detected in two types of gametes before mixing and cells 30min after mixing.

Molecular Cloning and Characterization of Two Genes Encoding gp138, a Cell Surface Glycoprotein Involved in the Sexual Cell Fusion of Dictyostelium discoideum

The sexual development of cellular slime molds is initiated by acquisition of sexual fusion competence of myxamoeboid cells. During the acquisition of fusion competence in NC4 and HM1 strain cells of Dictyostelium discoideum, opposite in mating types, a glycoprotein gp138, relevant to sexual cell fusion, is expressed on the cell surfaces of both the strains. To investigate the mechanisms controlling cell fusion, gp138 was purified and its N-terminal amino acid sequence was determined. An oligonucleotide with sequence predicted from the amino acid sequence was synthesized to isolate the gene encoding gp138. Two genes, referred to as GP138A and GP138B, were then cloned. They contain the region which encodes the N-terminal amino acid sequence of the gp138 protein and their structure is very similar on the whole. The C-terminal portion of the predictive polypeptides produced by the genes is highly hydrophobic and proline rich. It has homology with a portion of gp80, a glycoprotein for cell-cell adhesion, and PsA, a glycoprotein specific for prespore cells, reported previously in D. discoideum. The expression of GP138A is greater than that of GP138B. The mRNA of GP138A is expressed at the time of acquisition of fusion competence of cells during cultivation but the mRNA of GP138B is not.

Induction of Heterothallic and Homothallic Zygotes in Dictyostelium discoideum by Ethylene.

Dictyostelium mucoroides-7 (Dm7) and a mutant (MF1) derived from it exhibit homothallic macrocyst formation in the sexual process. As previously shown, the zygote formation during macrocyst formation is induced by a potent plant hormone, ethylene. The present work was undertaken to know if ethylene is also involved in heterothallic and homothallic macrocyst formation in D. discoideum. In heterothallic macrocyst formation between NC4 and V12M2 cells, ethionine, an analogue of methionine, inhibits macrocyst formation through arresting specifically the acquisition process of fusion competence. Such an inhibitory effect of ethionine was almost completely cancelled by co-application of ACC (1-aminocyclopropane-1-carboxylic acid), the immediate precursor of ethylene. Essentially the same effects of ethionine and ACC were also noticed on homothallic macrocyst formation in D. discoideum AC4. Thus it seems most likely that ethylene is required for the acquisition of fusion competence during macrocyst formation, and that in a variety of strains examined there is a common mechanism regulated by ethylene, beyond the difference of sexual modes.

Fusion-inhibiting monoclonal antibodies and their relevant antigens in relation to sexual process of Dictyostelium discoideum

Abstract Sexual cell fusion occurs between NC4 and HM1, the heterothallic strains in Dictyostelium discoideum. Cells of these strains are fusion incompetent when cultured on agar plates in the light and become fusion competent upon cultivation in a liquid medium in darkness. Two cell-surface components, gp70 and gp138, have been identified and characterized as being relevant to sexual cell fusion. Both are glycoproteins, and the former is detected only in fusion-competent HM1 cells, while the latter is detected both in fusion-competent HM1 and fusion-competent NC4 cells. We therefore suspect gp70 to be responsible for cell recognition and gp138, for membrane fusion. Therefore, NC4 cells are expected to possess specific surface molecule(s) that can be recognized by HM1 cells. In the present study, we raised monoclonal antibodies (mAbs) against membrane fractions of NC4 cells and selected fusion-inhibiting mAbs to identify novel molecules related to sexual cell fusion in D. discoideum. Out of the five mAbs we obtained three, DEI, GG6, and HH9, were characterized. DEI recognized antigens that specifically existed in fusion-competent NC4 cells but not in fusion-incompetent NC4 or HM1 cells. GG6 recognized cell-surface proteins with approximate molecular weights of 125 and 32 kDa in both fusion-competent NC4 and fusion-competent HMI cells. In addition GG6 also recognised other proteins commonly present in fusion-incompetent cells. The 125 kDa protein appeared to be the same as gp138. The epitope recognized by HH9 was sodium dodecyl sulfate (SDS)-sensitive. In an immunoblot of nondenatured polyacrylamide-gel electrophoresis (PAGE), HH9 detected two protein bands that were common to fusion-competent NC4 and fusion-competent HMI cells, but were distinct from gp138. These results suggest that other protein species in addition to gp138 are involved in sexual cell fusion between NC4 and HMI cells, with one having the properties of putative recognition molecules.

Correlation of Axenic Linkage Groups with the Position of the Microtubule-Organizing Center in Aggregating Dictyostelium.

Positioning of the microtubule-organizing center (MTOC) in Dictyostelium discoideum was found to be genetically regulated. We examined the wild-type strain NC-4 cells independently maintained in different laboratories, freshly recovered cells from spores stocked for over 20 years, the temperature-sensitive growth mutant HU49 isolated from NC-4, as well as strain V-12 which is the opposite mating-type to NC-4. During aggregation on nonnutrient agar plates, all these strains showed similar cell polarity, as defined by the alignment of the nucleus ahead of the MTOC. By contrast, in Ax2 and Ax3, axenic strains carrying axenic mutations on linkage groups II and III, the MTOC was usually positioned ahead of the nucleus. Cells containing axenic linkage group II but not III positioned the MTOC ahead of the nucleus. Conversely cell polarity of strains including axenic linkage group III but not II was similar to that of wild-type cells. Thus axenic linkage group II, probably axeC or other linked gene(s) not yet identified, is responsible for the location of the MTOC anterior to the nucleus during aggregation. The anterior positioning of the MTOCs was prevented by growth on bacteria in cells carrying both axenic linkage groups, but not in those carrying only axenic linkage group II.

Induction of gene expression in Dictyostelium by prestarvation factor, a factor secreted by growing cells

During growth, Dictyostelium cells continuously secrete a factor, PSF, that accumulates in proportion to cell density. At sufficient concentration, it triggers the production of discoidin I and certain lysosomal enzymes. Our earlier studies demonstrated these effects of PSF on protein and enzyme levels [Clarke et al., Differentiation 34:79-87, 1987; Clarke et al., Dev Genet 9: 315-326, 1988]. In the present study, we have examined whether PSF induces increased mRNA levels. By Northern blot analysis, we have found that discoidin I mRNA accumulates in exponentially growing NC4 cells as the cells reach high density; significant levels of mRNA are detectable in cells growing either on plates or in suspension, beginning about four generations before the end of exponential growth. High levels of discoidin I mRNA are also found in low-density cells grown in the presence of buffer conditioned by high-density cells. These results indicate that PSF induces the accumulation of discoidin I mRNA. Other "early developmental" genes, pCZ22 and the early I genes (16, 18, and 111), are also expressed in exponentially growing cells at high density or in the presence of conditioned buffer. We conclude that several genes previously found to be preferentially expressed very early in development are actually induced during late exponential growth by PSF.

Affinity purification of a 70K protein, a membrane protein relevant to sexual cell fusion in Dictyostelium discoideum

Sexual cell fusion occurs between HM1 and NC4, heterothallic strains in Dictyostelium discoideum. A membrane component of HM1 cells with a molecular mass of 70 kDa (70K protein) has been shown to be implicated in cell fusion (Urishihara et al. (1988) Cell Differ. Dev. 25, 81–88). In the present study, 70K protein was partially purified using affinity Sepharose on which membrane proteins of NC4 cells were immobilized. Through this process, involvement of Ca 2+ in the interaction of 70K protein with its receptor was suggested. Lectin staining of partially purified 70K protein indicated it to be a glycoprotein containing d-mannose and/or d-glucose residues.

Purification and characterization of gp138, a cell surface glycoprotein involved in the sexual cell fusion of Dictyostelium discoideum

Macrocyst formation in Dictyostelium discoideum is initiated by the fusion of cells between two opposite mating-type strains, such as NC-4 and HM1. When these cells become fusion-competent under appropriate environmental conditions, a specific protein involved in sexual cell fusion, 138 kDa, appears on the cell surface of both NC-4 and HM1 strains, as reported previously. The present study was carried out to purify and characterize this protein. The 138 kDa protein was shown to be a glycoprotein (gp138) which binds specifically to lectins, WGA, Con-A or LCA, but not to PNA, PHAE 4, RCA60 or RCA120. The isoelectric point of the gp138 was determined as pH 4.5-4.9. To confirm again the previous results, a Coomassie blue-stained gel containing the 138-kDa band was cut out following the SDS-polyacrylamide gel electrophoresis, and antiserum against this band protein(s) was obtained. Fab fragments of this antiserum caused the complete inhibition of sexual fusion between NC-4 and HM1 cells.

Identification of the cell surface molecule involved in sexual cell fusion of Dictyostelium discoideum

Abstract Dictyostelium discoideum was used as a model system for elucidating the molecular mechanism of sexual cell fusion. In heterothallic strains NC4 and HM1 of D. discoideum, complements in mating type, amoeboid cells acquire fusion competence only under certain environmental conditions, such as the presence of excess water and a certain period of darkness, to fuse sexually. The surface of cells which acquired fusion competence was found to possess specific antigens. Monovalent antibodies prepared from rabbit antiserum against fusion-competent NC4 cells inhibit the sexual cell fusion of these cells completely. Two specific antigenic proteins, 39 and 138 k Da in relative molecular mass and specific for fusion-competent cells, were detected. Only one, the 138-k Da protein, was capable of neutralizing the fusion-inhibitory activity of the monovalent antibody. These results show that the 139-k Da protein is the one involved in the sexual cell fusion of NC4 and HM1 strains in D. discoideum

Evidence that elevated intracellular cyclic AMP triggers spore maturation in Dictyostelium

ABSTRACT Spore maturation occurs during normal development in Dictyostelium when environmental influences induce a migrating slug to transform into a fruiting body. As the amoeboid prespore cells turn into refractile spores there is a burst of enzyme accumulation, including UDP-galactose epimerase, and at a later stage the exocytosis of preformed components of the spore coat. Evidence is presented here that this process is triggered by an elevated intracellular cAMP concentration. First, a number of rapidly developing (rde) mutants, whose cAMP metabolism had been investigated previously, are shown to be able to form spores in submerged monolayers, whereas wild-type strains are not. The phenotypes of these mutants are best explained by a derepression of the signal transduction pathway utilizing intracellular cAMP. Second and more direct, it is shown that the permeant cAMP analogues 8-Br-cAMP and 8-chlorophenylthio-cAMP, but not cAMP itself, can rapidly induce spore differentiation in wild-type amoebae incubated in submerged monolayers. These analogues also stimulate accumulation of UDP-galactose epimerase in slug cells transferred to shaken suspension. The ability to induce spore differentiation with Br-cAMP in wild-type strains provides a new technique that can be exploited in various ways. For instance, spore differentiation in strain V12M2 is induced by 8-Br-cAMP at very low cell densities, suggesting that neither cell contact nor additional soluble inducers are necessary in these conditions. In contrast NC4 cells may require an additional inducer. Spore differentiation is inhibited by the stalk-specific inducer DIF-1 suggesting that DIF-1 inhibits a target downstream of intracellular cAMP in the signal transduction pathway inducing spore differentiation.

A membrane protein with possible relevance to sexual cell fusion in Dictyostelium discoideum

The molecular mechanism of sexual cell fusion in Dictyostelium discoideum was studied using the heterothallic strains HM1 and NC4. Monovalent antibodies (Fab) prepared from rabbit antiserum against a crude membrane preparation of fusion-competent HM1 cells inhibited fusion between HM1 and NC4 cells. Six out of 43 antigenic proteins were found in fusion-competent HM1 cells but not in fusion-incompetent cells. Among them, only one protein with a molecular mass of 70 kDa was able to neutralize the fusion-inhibiting activity of Fab, suggesting its possible participation in sexual cell fusion.

Molecular cloning of cell-type-specific cDNAs exhibiting new types of developmental regulation in Dictyostelium discoideum

By screening cDNA libraries from NC-4 cells, we have obtained five prespore-specific and two prestalk-specific cDNA clones. The two classes of prespore genes began to be expressed at the tipped aggregate stage: one class was expressed throughout development, while the other was shut off during culmination. Another class of prespore genes was expressed in vegetative cells but only in prespore cells at the later stages. Prestalk-specific genes were first expressed at the tipped aggregate stage concurrently with the two prespore gene classes, indicating the importance of this stage for transcriptional regulation in both prestalk and prespore differentiation. Except for the first prespore gene class, these represent new cell-type-specific genes as to their temporal pattern of expression.

Cyclic AMP is an inhibitor of stalk cell differentiation in Dictyostelium discoideum

Cyclic AMP and DIF-1 (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)-1-hexanone) together induce stalk cell differentiation in vitro in Dictyostelium discoideum strain V12M2. The induction can proceed in two stages: in the first, cyclic AMP brings cells to a DIF-responsive state; in the second, DIF-1 alone can induce stalk cell formation. We report here that during the DIF-1-dependent stage, cyclic AMP is a potent inhibitor of stalk cell differentiation. Addition of cyclic AMP at this stage to V12M2 cells appreciably delays, but does not prevent, stalk cell formation. In contrast, stalk cell differentiation in the more common strain NC4 is completely suppressed by the continued presence of cyclic AMP. This fact explains earlier failures to induce stalk cells in vitro in NC4. We now consistently obtain efficient stalk cell induction in NC4 by removing cyclic AMP in the DIF-1-dependent stage. Cyclic AMP also inhibits the production of a stalk-specific protein (ST310) in both NC4 and a V12M2 derivative. Adenosine, a known antagonist of cyclic AMP action, does not relieve this inhibition by cyclic AMP and does not itself promote stalk cell formation. Finally, stalk cell differentiation of NC4 cells at low density appears to require factors in addition to cyclic AMP and DIF-1, but their nature is not yet known. The inhibition of stalk cell differentiation by cyclic AMP may be important in establishing the prestalk/prespore pattern during normal development, and in preventing the maturation of prestalk into stalk cells until culmination.

Analysis of the prestarvation response in growing cells of Dictyostelium discoideum.

We have previously shown that growing cells of Dictyostelium discoideum (strains NC4 and AX3) produce a soluble substance that accumulates in the medium in proportion to cell density; this substance regulates the production of certain proteins previously thought to be induced by starvation [Clarke et al., 1987]. We suggest the name PSF (prestarvation factor) for this substance. During growth, Dictyostelium cells monitor the relative concentrations of PSF and food bacteria. When PSF reaches a sufficiently high level relative to the concentration of bacteria, synthesis of PSF-regulated proteins is induced. We propose the name prestarvation response for this induction, which takes place in exponentially growing cells several generations before the food bacteria are depleted. We have explored the mechanism by which the food bacteria inhibit the response of Dictyostelium cells to PSF. We find that the bacteria do not inactivate PSF or inhibit its production; instead, they affect the ability of NC4 cells to detect PSF, possibly by binding to the same cell surface receptor. In the absence of bacteria, as during axenic growth of AX3 cells, the prestarvation response occurs at much lower cell densities, probably accounting for the presence of certain developmentally regulated mRNAs and proteins in axenic cultures.

Fusion of cell ghosts with intact cells in Dictyostelium discoideum: Differential response of opposite mating-type cells

Abstract The sexual cycle of Dictyostelium discoideum is initiated by the fusion of cells that are of opposite mating types (e.g. NC4- and HM1-type cells). Cells grown in light on agar plates are not capable of sexual cell fusion, but become capable when cultured in the dark in a liquid medium. Cells in the incapable state are called fusion-incompetent cells, and cells in the latter state, fusion-competent cells. To gain some understanding of the mechanism of cell fusion, cell ghosts prepared by freeze-thawing intact cells were incubated with intact cells. The cell ghosts killed the intact cells by directly fusing with them, the extent of fusion depending on the particular strains employed and the fusion-competency of the intact cells and of the cells from which the cell ghosts had been prepared. A detailed examination revealed that fusion-competent NC4 cells were always more easily killed by cell ghosts than fusion-incompetent NC4 cells. It also became apparent that cell ghosts prepared from fusion-competent NC4 cells killed all cell types far more efficiently than did those prepared from fusion-incompetent NC4 cells. However, fusion-competent and fusion-incompetent HM1 cells were equally sensitive to cell ghosts, and cell ghosts prepared from fusion-competent HM1 cells had the same ability to kill as those prepared from fusion-incompetent HM1 cells. From these findings, it thus appears that opposite mating-type cells have distinct membrane properties related to sexual cell fusion.

Fusion of cell ghosts with sexually opposite type cells in Dictyostelium discoideum

In the sexual cycle of Dictyostelium discoideum, haploid cells of two opposite mating types, strains HM1 and NC4, acquire fusion-competence under certain conditions, such as suspension culture in the dark, and fuse specifically to form giant zygote cells. Each giant cell engulfs the surrounding cells, gradually increases in size, and finally develops into a macrocyst that is a sexual structure in D. discoideum. Fusion-competent HM1 cells suspended in a solution were frozen and thawed to make cell ghosts. When cell ghosts were introduced into fusion-competent and -incompetent intact NC4 cells, the cell ghosts killed them in a short time, but the fusion-competent cells were killed in preference to the fusion-incompetent cells. This killing occurred through the fusion of the cell ghosts directly to intact cell membranes. Since the fusion was specific, the fusion between ghosts and cells appears to be essentially the same as that between intact cells during the sexual cycle in molecular mechanisms.

Axenic Growth of Dictyostelium discoideum Wild-type NC-4 Cells and its Relation to Endocytotic Ability

A method for the axenic growth of Dictyostelium discoideum wild-type strain NC-4 is described. There were some differences between the nutritional requirements of strain NC-4 and an axenic strain Ax-2 derived from NC-4. Of the numerous components in growth media, the nature and concentration of the peptone was especially critical for axenic growth of NC-4 cells; 3% Bacteriological-peptone (Oxoid) giving the best growth rate and yield. NC-4 cells seemed to require more vitamin B12 than Ax-2 cells. Other vitamins like folic acid, biotin, and riboflavin also stimulated axenic growth of NC-4 cells. Pinocytosis was examined by the use of FITC-dextran and the results showed that the above peptone considerably enhanced the pinocytotic activity of both NC-4 and Ax-2 cells. Bacto-peptone (Difco) also enhanced pinocytosis to a similar extent, though it never supported axenic growth of NC-4 cells. Therefore, stimulation of pinocytosis may be necessary for nutrient uptake, but is not sufficient for axenic growth. The pinocytotic ability of cells decreased conspicuously during the course of development, particularly just before aggregation. The biological significance of this is discussed in relation to cell differentiation.

Macrocyst development in Dictyostelium discoideum. III. Cell-fusion inducing factor secreted by giant cells.

A factor was discovered that markedly enhances the degree of fusion activity between cells of the opposite mating-type strains, MI1 and NC4, in the sexual cycle of Dictyostelium discoideum. This factor, designated cell-fusion inducing factor (CFIF), was detected initially in supernatants taken from 24 h dark-grown mixed cultures of HM1 and NC4 cells, and subsequently shown to be secreted by giant cells - the fusion products of HM1 and NC4 cells. HM1 cells, cultured in the dark at appropriate temperature, normally acquire fusion-competence specific to NC4 cells. The addition of CFIF to such dark-grown HM1 cultures results in a marked increase in their fusion-competence. In addition, when CFIF is added to light-grown HM1 cultures, in which cells normally do not acquire the ability to fuse with NC4 cells, fusion-competence is induced. The fusion-competence of NC4 cells is unaffected by CFIF, being quite high under the culture conditions used here. Experiments, using actinomycin D, daunomycin and cycloheximide showed that the secretion of CFIF from giant cells requires synthesis of RNA and protein. Possibly, cell fusion triggers production of CFIF, which is rapidly released into the external medium.

Macrocyst development in Dictyostelium discoideum. II. Mating-type-specific cell fusion and acquisition of fusion-competence.

The early events of macrocyst development in Dictyostelium discoideum have been investigated using a new culturing system. When cells of opposite mating-types, HM1 and NC4, are cultured together at the appropriate temperature in the dark, giant cells appear, ingest the surrounding amoebae, and develop into macrocysts. Although these giant cells have been assumed to be the products of the fusion of opposite mating-type cells, no experimental evidence to prove this assumption has been obtained using such mixed-culture systems. In order to avoid the complexities involved in mixed-culturing, we have developed a new system involving the separate culture, and later mixing, of opposite mating-type cells. This has enabled us to obtain direct evidence that giant cells are produced by fusion between opposite mating-type cells. Cell fusion occurs immediately after mixing and is completed within 30 min. As a number of cells fuse simultaneously, giant cells produced by this method are very large multinucleated cells, and not binucleated zygotes. Using this system we also discovered the following facts related to giant cell formation: (1) cells can acquire their fusion competence without the presence of cells of the opposite mating-type; (2) only HM1 cells require darkness to acquire their fusion competence; (3) the restrictive temperature, 25 degrees C, inhibits the induction of fusion competence in HM1 cells, but not in NC4 cells.

Induction of Macrocyst Formation by Factors Secreted by Giant Cells in Dictyostelium discoideum: (D. discoideum/sexual cycle/induction of macrocyst formation).

The fusion of cells of complementary mating types to produce giant cells has been shown to be the critical event to induce macrocyst formation in Dictyostelium discoideum. We have examined the way in which giant cells use diffusible factors to influence the developmental mode of nearby cells using an experimental design in which NC4 cells are allowed to develop on a dialysis membrane above a suspension of giant cells. We have observed that giant cells are able to inhibit independent aggregation and stream formation in the upper cells and become the dominant aggregation centers. In addition giant cells are able to redirect local amoeba away from the fruiting-body and toward the macrocyst mode of development. We show that these effects are mediated by diffusible factors of under 2,000 MW. and discuss possible mechanisms of action.