The regulation of membrane fusion during sexual development in Dictyostelium discoideum

During the first 24 h of sexual development in Dictyostelium discoideum, three sequential events of membrane fusion occur: gamete fusion, pronuclear fusion, and phagocytosis. The early events of sexual development are regulated by a diverse group of endogenous molecules: (i) a volatile sexual pheromone, (ii) a protein cell fusion inducing factor (CFIF), (iii) a low molecular weight autoinhibitor, (iv) and cyclic AMP. CFIC enhances cell fusion while the autoinhibitor and cyclic AMP both inhibit the event. Both extracellular and intracellular calcium ions are essential for cell and pronuclear fusion. Pharmacological analyses show that the intracellular functions of the divalent cation in these processes are mediated by calmodulin. The autoinhibitor appears to function by inhibiting calmodulin activity. Glucose, mannose, and N-acetylglucosamine containing glycoproteins have been shown to function in both cell fusion and phagocytosis. The interplay between all of these diverse molecules is examined and a review of all of the recent literature is presented.     

Aggregation during sexual development in Dictyostelium discoideum.

A microcinematographic analysis of the behaviour and movements of cells and cell masses in mated cultures (NC4 X VI2) of Dictyostelium discoideum indicates that a chemotactic process directs cell aggregation during macrocyst development. Zygote giant cells form before aggregation begins and act as the aggregation centres. Young multicellular macrocyst stages are sources of cyclic AMP, and amoebae from macrocyst cultures orient chemotactically to cyclic AMP. The data, coupled with other characteristics such as pulsatile streaming, suggest that the aggregation process leading to macrycyst development is the same as that occurring during fruit construction. Other aspects of sexual development are also discussed. Based upon these data, we propose a model for the sequence of events leading to macrocyst development in D. discoideum.     

Phagocytic specificity during sexual development in Dictyostelium discoideum

During the sexual cycle of Dictyostelium discoideum, zygote giant cells develop and serve as foci for further development by chemoattracting and cannibalizing hundreds of local amoebae. Previous work has shown that the phagocytic process bears similarities to and differences from asexual endocytosis. In the present study, sexual phagocytosis in D. discoideum was found to be species and developmental stage specific. It was inhibited selectively by glucose and concanavalin A. Although a partial, inhibitory effect of mannose on phagocytosis was not statistically significant, alpha-methylmannosamine, like alpha-methyl-glucose, significantly restored the phagocytic competence of giant cells treated with concanavalin A. Other sugars (N-acetyl-glucosamine, N-acetylgalactosamine, and galactose) and lectins (wheat germ agglutinin, Ulex europus type I, and Ricinis communis agglutinin type I) had no significant effect on sexual phagocytosis. Together these data indicate that a glucose-type receptor is involved in selective uptake of D. discoideum amoebae by giant cells.     

Ca2+ and cell fusion during sexual development in liquid cultures of Dictyostelium discoideum

Cell fusion resulting in zygote giant cell formation is the first observable event of sexual development in D. discoideum. The results reported here show that this process is Ca²⁺-dependent and that by increasing the level of Ca²⁺ in the medium the number of cell fusions can be increased 57-fold over control cultures. The data also suggest that Ca²⁺ has both an early and late function in the development of zygotes and these functions are mediated at the cell surface. These results plus the availability of a liquid culture for generating large volumes of cells make sexual development in D. discoideum an excellent system for the analysis of membrane fusion in eukaryotes.     

Evidence for chemotaxis during sexual development in Dictyostelium discoideum.

Amoebae in mated cultures of Dictyostelium discoideum show oriented movement towards young aggregates, suggesting that cemotaxis is involved in macrocyst development. Amoebae also show directional movement towards midendocyte stages, indicating that as the macrocyst develops it continues to be a source of chemoattractant. These data are discussed in terms of our current knowledge about mating in the cellular slime moulds.     

Pheromones do regulate sexual development in Dictyostelium discoideum

Based on negative data, Douglas et al refuted earlier work by the O’Day laboratory on the role of pheromones in the sexual development of Dictyostelium discoideum. This letter addresses some of the issues with their study.     

A novel cyclic AMP metabolism exhibited by giant cells and its possible role in the sexual development of Dictyostelium discoideum

In Dictyostelium discoideum cyclic AMP (cAMP) metabolism during macrocyst development, i.e., the sexual cycle of this organism, and in giant cells, i.e., fusion products from opposite mating-type cells, was investigated. The pattern of change in cAMP levels during macrocyst development differed considerably from that observed during fruiting-body formation, i.e., the asexual cycle. Giant cells produced and excreted considerable amounts of cAMP. Adenylate cyclase activity catalyzing cAMP production in giant cells was comparable to that of unfused cells. However, the activity of membrane-bound phosphodiesterase in giant cells was extremely low, and no extracellular phosphodiesterase was excreted. A phosphodiesterase inhibitory protein was secreted in excess by giant cells.     

Synthesis of specific proteins for sexual development in Dictyostelium discoideum

The development of Dictyostelium discoideum may proceed by two pathways, macrocyst or fruiting-body formation, the former being the sexual and the latter the asexual cycle. The pathway of development depends on the presence or absence of zygote giant cells which are produced through fusion of opposite mating-type cells in a population, in heterothallic strains. During the early stages of macrocyst development the patterns of developmentally regulated proteins were noted to differ considerably from those during fruiting-body development. Furthermore, the haploid cells around zygote giant cells synthesized a large number of specific proteins for macrocyst development through the influence of giant cells.     

Zygote giant cell differentiation in Dictyostelium discoideum: biochemical markers of specific stages of sexual development.

Sexual development in Dictyostelium discoideum has many unique features making it an attractive eukaryotic model system for the study of biomembrane fusion and intercellular communication. The work presented here provides primary biochemical evidence for two distinct phases during early sexual development that appear to be defined by calcium-dependent gamete cell fusion. In addition, we introduce a novel procedure for the enrichment of zygote giant cells and use this method to define certain wheat-germ agglutinin binding glycoproteins which are specifically located in zygote giant cells and others which are markers for surrounding amoebae in the second phase of development. In addition, a G protein which is present in high amounts early in development is unique to giant cells in the second phase, suggesting a role in phagocytosis. Finally, alkaline phosphatase activity was found to mark the first phase of sexual development, suggesting a role in cell fusion. This contrasts with the patterns of alpha-mannosidase and beta-glucosidase activity that increase late in the second developmental phase, where they likely function in endocyte digestion during the cytophagic period. The developmental significance of these findings is discussed.     

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

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.     

Gamete fusion and cytokinesis preceding zygote establishment in the sexual process of Dictyostelium discoideum

Cells of Dictyostelium discoideum become sexually mature under submerged and dark conditions, and fuse with opposite mating-type cells to form zygote giant cells, which gather surrounding cells and finally develop into dormant structures called macrocysts. In the present study, we found that the multinuclear fused cells formed during this process frequently underwent cytokinesis driven by random local movements. The split cells were capable of re-fusion, and repeated cytokinesis. These radical behaviors continued until the extensive cell aggregation started around the giant cells. Thus, gamete fusion and initiation of zygote development do not coincide in the mating of D. discoideum. Analyses by confocal microscopy and flow cytometry indicated that the cessation of the random movement followed pronuclear fusion, and that microtubule organizing centers (MTOC), abundant in the fused cells at the beginning, gradually decreased and only one of them remained within the developed macrocyst. Some of the genes known to control cell movement, such as rasGEFB and rasS, increased shortly before the cessation of repeated fusion-cytokinesis and initiation of phagocytosis. These results suggest that the sequential molecular events are necessary in D. discoideum after gamete fusion to establish a new individuality of zygotes.     

Changes in phospholipid composition during the development of Dictyostelium discoideum

The phospholipid composition of Dictyostelium discoideum cells was determined at various stages of development by two-dimensional, thin-layer chromatography and reaction thin-layer chromatography. Major phospholipids of D. discoideum which were detectable throughout all stages of development were ethanolamine phosphoglyceride and choline phosphoglyceride. Ethanolamine phosphoglyceride and choline phosphoglyceride were found as their plasmalogen forms at 45–58 and 10–24%, respectively. There were no qualitative changes in phospholipid composition during the development, but quantitative changes did occur. The relative content of ethanolamine phosphoglyceride in the total phospholipids gradually decreased from 60% at the vegetative stage to 44% at the 1-day-sorocarp stage. In contrast, choline phosphoglyceride gradually increased from 27% at the vegetative stage to 48% at the preculmination stage, and then gradually decreased to 43% during the culmination. The decrease in ethanolamine phosphoglyceride content during the middle and late development was due mainly to the decreased amount of its plasmalogen form but the increase of choline phosphoglyceride was independent of quantitative changes of its plasmalogen form. Other minor components of phospholipid did not show significant changes in their levels. The causes of these changes in contents of ethanolamine phosphoglyceride and choline phosphoglyceride were examined by label and chase experiments with [³H]ethanolamine and [¹⁴C]choline. It seems that one-third to one-half of the increased amount of choline phosphoglyceride was due to stepwise methylation of ethanolamine phosphoglyceride, and the remaining two-thirds to one-half was caused by de novo synthesis of choline phosphoglyceride from CDP-choline and diglyceride.     

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.     

A mutation in the cAMP signaling pathway affects sexual development of Dictyostelium discoideum

Amoebae of cellular slime molds have two developmental modes, asexual fruiting body formation and sexual macrocyst formation. How developmental choice is made is an interesting subject of wide importance. Light exposure and dry conditions are favorable for asexual development, while conditions of darkness and high humidity are so for sexual development. In Dictyostelium discoideum , the latter conditions enhance zygote formation, which determines the fate of surrounding cells for sexual development. Here, a mutant (TMC1) defective in the post-fusion aggregation of cells during sexual development is described. This mutant is also aggregationless in asexual development, and the level of cyclic adenosine monophosphate (cAMP) receptor is reduced. Correspondingly, a series of existing mutants with defects in cAMP signaling pathways showed the same sexual phenotype as TMC1. These results suggest that molecular mechanisms of development are shared by the two alternative developmental modes.     

Genes involved in Dictyostelium discoideum sexual reproduction

Macrocyst formation in the cellular slime moulds is a sexual process induced under dark and humid conditions. Normal development life cycle in these organisms involves proliferation by cell division and, upon starvation, formation of multicellular aggregates and fruiting bodies, consisting of spores and stalk cells. Macrocyst formation, cell division by binary fission and spore formation are thus three alternative modes of reproduction, for which it is of interest to understand how a choice is made. The genetic basis of asexual development and fruiting body formation is well known, by contrast information on the genetic control of sexual reproduction during macrocyst formation is scarce. In Dictyostelium discoideum, the most widely used species, several cell-surface proteins relevant to sexual cell fusion have been identified using cell fusion-blocking antibodies, but isolation of the relevant genes has been unsuccessful. Analysis of sexually deficient mutants, some of which are normal for asexual development, has shown that sexual reproduction is regulated by both specific genes and genes that are also involved in asexual development. Reverse genetic analysis of 24 genes highly enriched in a gamete-specific subtraction library has revealed four genes involved in the regulation of sexual cell interactions. One of them was found to be a novel regulator of the cAMP signalling pathway specific to sexual development. Studies on the molecular genetic control of the sexual cycle will be reviewed and their contribution to our understanding of the organization and function of the D. discoideum genome as a whole discussed.     

Small GTPase RacF2 affects sexual cell fusion and asexual development in Dictyostelium discoideum through the regulation of cell adhesion

Cells of Dictyostelium discoideum become sexually mature when submerged and in darkness, and fuse with opposite mating-type cells as gametes. The gene for a Rho GTPase, RacF2, is one of the extremely gamete-enriched genes (>100-fold) identified by us previously. Here, we isolated knockout, overexpression, constitutively active and dominant negative mutants of RacF2, and analyzed their phenotypes. These mutants showed anomalies in the extent of sexual cell fusion and asexual development as well as in EDTA-sensitive cell-cell adhesion. It is suggested that RacF2 controls the process of sexual and asexual development through the regulation of cellular adhesiveness. An analysis of the expression of all 18 rac family genes by real-time polymerase chain reaction revealed that four additional genes, rac1b, rac1c, racF1 and racG, were induced during maturation, suggesting their possible involvement in sexual cell interactions.     

The phosphorylation of membranal proteins in Dictyostelium discoideum during development

The pattern of membranal phosphoproteins in Dictyostelium discoideum changes during development (D. S. Coffman, B. H. Leichtling, and H. V. Rickenberg, 1981, J. Supramol. Struct. Cell. Biochem. 15, 369–385). Phosphorylation of six membranal proteins occurred concomitantly with their synthesis. Cyclic AMP stimulated the precocious synthesis of a phosphoprotein, of molecular weight 80,000, which corresponds to contact sites A. Phosphoserine was the only phosphorylated amino acid found in the five phosphoproteins examined. In at least two phosphoproteins, that corresponding to contact sites A and a phosphoprotein of molecular weight 64,000, the phosphate moiety did not turn over.     

Localization of adenylate cyclase during development of Dictyostelium discoideum

Cyclic AMP is known to function as the chemotactic signal during aggregation of single-celled amoebae of the cellular slime mold Dictyostelium discoideum. Evidence from several laboratories has accumulated suggesting that cAMP also acts as a regulatory molecule during Dictyostelium multicellular differentiation. We have used ultramicrotechniques and a sensitive radioimmunoassay in the localization of adenylate cyclase, the cAMP synthetic enzyme, during the development of Dictyostelium. We demonstrate that adenylate cyclase activity is localized in the prespore cells of the culminating individual with no activity detectable in the prestalk region. We show that this lack of activity in the stalk may be due to a masking by an endogenous inhibitor of the enzyme. Within the spore mass we found an increasing gradient of enzyme activity toward the base. These data, along with that from the localization of cyclic nucleotide phosphodiesterase, indicate that an enzymatic potential exists for the creation of cAMP gradients during development in the organism. Such a gradient may provide positional information necessary to direct the terminal differentiation of spore and stalk cells.     

Localization of adenylate cyclase during development of Dictyostelium discoideum

Abstract. Cyclic AMP is known to function as the chemo-tactic signal during aggregation of single-celled amoebae of the cellular slime mold Dictyosteliwn discoideum. Evidence from several laboratories has accumulated suggesting that cAMP also acts as a regulatory molecule during Dictyostelium multicellular differentiation. We have used ultra-microtechniques and a sensitive radioimmunoassay in the localization of adenylate cyclase, the cAMP synthetic enzyme, during the development of Dictyostelium. We demonstrate that adenylate cyclase activity is localized in the pre-spore cells of the culminating individual with no activity detectable in the prestalk region. We show that this lack of activity in the stalk may be due to a masking by an endogenous inhibitor of the enzyme. Within the spore mass we found an increasing gradient of enzyme activity toward the base. These data, along with that from the localization of cyclic nucleotide phosphodiesterase, indicate that an enzymatic potential exists for the creation of cAMP gradients during development in the organism. Such a gradient may provide positional information necessary to direct the terminal differentiation of spore and stalk cells.     

Distribution of cAMP-dependent protein kinase during development in Dictyostelium discoideum

During the developmental cycle of Dictyostelium discoideum cyclic AMP functions as both a chemotactic signal for aggregation and a regulatory molecule during later events of differentiation. Morphological and biochemical data suggest that cAMP may direct cells during morphogenesis and differentiation. We utilized microtechniques to determine the stage- and cell-specific levels of the cAMP-dependent protein kinase, the probable intracellular cAMP receptor. Kinase activity was low and non-cAMP-dependent in amoebae and early aggregates but increased and became cAMP-dependent in aggregates after the formation of tight cell contacts. Maximum kinase activity and cAMP dependency occurred during the slug and culmination stages. The only differential distribution of the kinase within a single stage occurred during culmination when the activity in the stalks was approximately one-fourth of that in the prespore mass. Preliminary evidence indicates that this difference is not due to an inhibitor. In all other stages tested cAMP-dependent protein kinase activity was equal in prespore and prestalk cells.