Ca 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.     

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.     

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.     

Nuclear fusion in multinucleated giant cells during the sexual development of Dictyostelium discoideum

Macrocyst development in Dictyostelium discoideum, is generally considered a sexual phase. This development is initiated by the formation of a giant cell, the result of the fusion of two different mating type haploid cells, such as NC4 and HM1. The giant cell engulfs unfused surrounding cells to develop into a macrocyst. Therefore, if the macrocyst is a sexual structure, the giant cell must be a diploid zygote. However, under certain conditions, a very large multinucleated giant cell containing several dozens of nuclei is formed, followed by normal development into a macrocyst. In such a multinucleated giant cell, it was found that only two nuclei fuse together to produce a diploid zygote and all others disappear at the early stage of development. The diploid nucleus undergoes meiosis and subsequently subdivides into a number of haploid progeny cells later released from the macrocyst to initiate new life cycles.     

Signalling and sex in the social amoebozoans

The social amoebozoans have a life tricycle consisting of asexual multicellular development leading to fruiting bodies, sexual multicellular development resulting in macrocysts, and unicellular development generating microcysts. This review covers the events of sexual development in the best‐studied heterothallic (Dictyostelium discoideum) and homothallic (D. mucoroides) mating systems. Sexual development begins with pheromonal interactions that produce fusion‐competent cells (gametes) which undergo cell and pronuclear fusion. Calcium‐ and calmodulin‐mediated signalling mediates these early events. As they initiate chemotactic signalling, each zygote increases in size becoming a zygote giant cell. Using cyclic AMP (cAMP), the zygote chemotactically lures in amoebae and engulfs them in an act of cannibalistic phagocytosis. Chemotaxis proceeds more quickly than endocytosis because the breakdown products of cAMP (5‐AMP, adenosine) bind to a presumptive adenosine receptor to inhibit sexual phagocytosis. This slowing of phagocytosis allows amoebae to accumulate around the zygote to form a precyst aggregate. Zygote giant cells also produce several other signalling molecules that feed back to regulate early events. The amoebae surrounding the zygote seal their fate as zygotic foodstuff by secreting a primary cellulose wall, the extracellular sheath, around the zygote and aggregated amoebae, which prevents their escape. Phagocytosis within this precyst continues until all peripheral amoebae are internalized as endocytes and the final macrocyst wall is formed. Endocyte digestion results in a mature macrocyst with a uniform cytoplasm containing a diploid nucleus. After detailing the morphological events of heterothallic and homothallic mating, we review the various intercellular signalling events and other mechanisms involved in each stage. This complete and comprehensive review sets the stage for future research on the unique events that characterize sex in the social amoebozoans.     

Cell fusion, nuclear fusion, and zygote differentiation during sexual development of Dictyostelium discoideum

The fluorescent nuclear stain Hoechst 33258 was used to study the nuclear events during mating of Dictyostelium discoideum in liquid culture. These studies revealed that cell fusion begins about 11 hr after the sexually compatible cultures are mixed and continues until 26 hr. Approximately 37% of the cells fuse during this 15-hr period. At first the fused cells are relatively small, but by 20 hr the fusion products become evident as morphologically distinct giant cells. Starting at 22 hr these giant cells are transformed into true zygotes as nuclear fusion begins. Both the fusion of amebae and the differentiation of zygote giant cells are Ca²⁺-dependent events as revealed by studies using EGTA. The nuclear events of zygote differentiation involve nuclear swelling, migration, and fusion. The precise timing of these events has been detailed. Of particular interest for genetic analyses via the macrocyst is the presence of a small population of multinucleate cells (maximum level is 1.67% of the cell population) which usually possess 3 or 4 nuclei but may have as many as 10 or more. Although these multinucleate cells contain many nuclei, our evidence suggests that only one is a zygote nucleus. The genetic implications of these data and the potential value of using the mating system for the analysis of cell fusion are discussed.     

Social amoebae mating types do not invest unequally in sexual offspring

Unequal investment by different sexes in their progeny is common and includes differential investment in the zygote and differential care of the young. The social amoeba Dictyostelium discoideum has a sexual stage in which isogamous cells of any two of the three mating types fuse to form a zygote which then attracts hundreds of other cells to the macrocyst. The latter cells are cannibalized and so make no genetic contribution to reproduction. Previous literature suggests that this sacrifice may be induced in cells of one mating type by cells of another, resulting in a higher than expected production of macrocysts when the inducing type is rare and giving a reproductive advantage to this social cheat. We tested this hypothesis in eight trios of field‐collected clones of each of the three D. discoideum mating types by measuring macrocyst production at different pairwise frequencies. We found evidence that supported differential contribution in only two of the 24 clone pairs, so this pattern is rare and clone‐specific. In general, we did not reject the hypothesis that the mating types contribute cells relative to their proportion in the population. We also found a significant quadratic relationship between partner frequency and macrocyst production, suggesting that when one clone is rare, macrocyst production is limited by partner availability. We were also unable to replicate previous findings that macrocyst production could be induced in the absence of a compatible mating partner. Overall, mating type‐specific differential investment during sex is unlikely in microbial eukaryotes like D. discoideum.     

Environmental factors inducing sexual development inDictyostelium discoideum

In the heterothallic strains NC4 and HM1 ofDictyostelium discoideum, sexual development is initiated by the formation of diploid zygotic giant cells produced through the fusion of these two opposite mating-type haploid cells. For sexual cell fusion, amoeboid cells must first acquire fusion competence, which requires culture under certain environmental conditions, such as darkness, excessive water, and sufficient bacteria as food. However, in the subsequent stages of cell fusion and development of the giant cells into mature macrocysts, cells do not require the above conditions. Cell fusion and development into macrocysts were able to occur even in light with minimum water and in the absence of bacteria. For cell fusion calcium ions were required.     

ENZYMATIC DISSOCIATION OF NASCENT MACROCYSTS AND PARTITION OF THE LIBERATED CYTOPHAGIC GIANT CELLS IN DICTYOSTELIUM MUCOROIDES*

Nascent macrocysts of the cellular slime mold Dictyostelium mucoroides were dissociated enzymatically and the liberated cytophagic giant cells were partitioned by dextrin density gradient centrifugation. Enzymatic and cytochemical studies revealed that the primary wall is composed mainly of cellulose (β-1,4-glucan) associated with polysaccharides including hemicellulose, pectic substances and á-1,4-glucan. The buoyant density of the liberated cytophagic giant cells and peripheral cells was determined by density gradient centrifugation, and partitioning of the cells was possible due to the difference in this property. The process of macrocyst reconstitution was investigated using dissociated cells. The isolated cytophagic giant cell has a specific affinity for other cytophagic giant cells and predominantly ingests them by phagocytosis, while it retains the ability to ingest peripheral cells. The present study provides a clue for investigating the differentiation and development of sexual cells, since only the cytophagic giant cell gives rise to a zygote in macrocyst formation.     

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.     

High density cultivation of Dictyostelium discoideum in a rotating polyurethane foam-bed bioreactor

The production of recombinant glycoproteins in Dictyostelium discoideum by conventional cell culture methods was limited by low cell density as well as low growth rate. In order to achieve high cell density cultivation, polyurethane foam (PUF) with high porosity was introduced as new matrix for the immobilization of D. discoideum. The results showed that about 88–93% cells of D. discoideum were adsorbed onto the PUF particles after 100 min equilibrium between adsorbed and free cells, and the highest immobilization rate was achieved by adding the same quantity of PUF matrix with the thin cylinder style. Furthermore, polyurethane foam was used as the immobilization matrix in a rotating PUF-bed bioreactor system. With batch cultures in the rotating bed bioreactor, the concentration of immobilized cells in the PUF carrier increased to 4.2 × 10⁷ cells ml⁻¹ after 167 h cultivation, which was about fourfold higher than the maximal cell density in the conventional free-cell culture. Further studies showed that the cells of D. discoideum were not just simply adsorbed on the surfaces, but actively attached to the surfaces through their network of pseudopodia or filopodia. The present work is very promising to improve the productivity of recombinant proteins in D. discoideum with high cell density in this novel rotating bed bioreactor.     

Cell Fusion Promoting Factor Common to Homothallic and Heterothallic Mating Systems in Dictyostelium discoideum1

Cellular slime mould Dictyostelium discoideum propagates as single haploid cells and under certain environmental conditions enters into a sexual cycle called macrocyst formation. There are homothallic and heterothallic strains reported, the former being able to form macrocysts in clonal cell populations while the latter to do so only in the presence of opposite mating-type strains. Molecular basis for differential mating systems is an intersting subject totally unknown yet. In the present study, sexual cell interactions in AC4, a homothallic strain of D. discoideum, was studied in comparison with the heterothallic mating system. The conditoned medium of AC4 cells was found to promote the sexual cell fusion among themselves. In addition, it also enhanced the cell fusion between heterothallic strains. Furthermore, the conditioned medium obtained from the mated culture of heterothallic strains reported to induce the sexual cell fusion in the heterothallic strains (Saga and Yanagisawa, 1983) was found also to promote the cell fusion in AC4. These results suggest that common regulatory mechanisms operate for sexual cell fusion among different mating systems in D. discoideum.     

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.     

Properties of cell surface carbohydrates in sexual reproduction of the Closterium peracerosum–strigosum–littorale complex (Zygnematophyceae,Charophyta)

The Closterium peracerosum–strigosum–littorale complex is a best characterized zygnematophycean green alga with respect to the process of sexual reproduction. Intercellular communication mediated by two sex pheromones has been well documented in this organism, but information concerning direct cell–cell recognition and fusion of cells involved in conjugation processes has not yet been clarified. In this study, we examined the properties of cell surface carbohydrates in vegetative and reproductive cells using a variety of fluorescein isothiocyanate labeled lectins as probes. Among 20 lectins tested, 10 bound to the Closterium cell surface, and eight of these were specific for the cells involved in sexual reproduction. In addition, some of the lectins inhibited the progress of zygote formation. In particular, Lycopersicon esculentum lectin (LEL) and ConcanavalinA (ConA) considerably inhibited zygote formation (23.6% and 0% of zygotes formed, respectively, compared with the control). LEL mainly accumulated on conjugation papillae and on the surface and lumens of empty cell walls remaining after zygote formation. ConA bound to both vegetative and sexually reproductive cells and strongly accumulated on the conjugation papillae of the latter, indicating ConA binding material(s) are non‐specifically present in Closterium cells but some of the material(s) would be essential for zygote formation. These results suggest that different carbohydrates specifically recognized by these lectins are involved in cell recognition and/or fusion during conjugation processes in the C. psl. complex.     

Evidence for somatic embryogenesis during plant regeneration from seedling-derived callus of dodder (<Emphasis Type="Italic">Cuscuta trifolii</Emphasis> Bab. et Gibs)

 In this paper comparative histological studies of embryo-like structures originating from callus cultures, and zygotic embryos originating from sexual seeds of Cuscuta trifolii are reported. The embryos of somatic cell and zygote origin showed similar morphological and anatomical features, such as a complete lack of cotyledon development and the differentiation of a developmentally unique root primordium specialised for water storage. Based on these findings, the regeneration of C. trifolii from callus cultures is shown to proceed along the pathway of somatic embryogenesis. Received: 9 November 1998 / Revision received: 22 April 1999 / Accepted: 29 June 1999     

THE SEXUAL LIFE CYCLES OF PFIESTERIA PISCICIDA AND CRYPTOPERIDINIOPSOIDS (DINOPHYCEAE)1

Sexual life cycle events in Pfiesteria piscicida and cryptoperidiniopsoid heterotrophic dinoflagellates were determined by following the development of isolated gamete pairs in single‐drop microcultures with cryptophyte prey. Under these conditions, the observed sequence of zygote formation, development, and postzygotic divisions was similar in these dinoflagellates. Fusion of motile gamete pairs each produced a rapidly swimming uninucleate planozygote with two longitudinal flagella. Planozygotes enlarged as they fed repeatedly on cryptophytes. In <12 h in most cases, each planozygote formed a transparent‐walled nonmotile cell (cyst) with a single nucleus. Zygotic cysts did not exhibit dormancy under these conditions. In each taxon, dramatic swirling chromosome movements (nuclear cyclosis) were found in zygote nuclei before division. In P. piscicida, nuclear cyclosis occurred in the zygotic cyst or apparently earlier in the planozygote. In the cryptoperidiniopsoids, nuclear cyclosis occurred inthe zygotic cyst. After nuclear cyclosis, a single cell division occurred in P. piscicida and cryptoperidiniopsoid zygotic cysts, producing two offspring that emerged as biflagellated cells. These two flagellated cells typically swam for hours and fed on cryptophytes before encysting. A single cell division in these cysts produced two biflagellated offspring that also fed before encysting for further reproduction. This sequence of zygote development and postzygotic divisions typically was completed within 24 h and was confirmed in examples from different isolates of each taxon. Some aspects of the P. piscicida sexual life cycle determined here differed from previous reports.     

Aplanogamous sexual reproduction in Carteria eugametos (Volvocales,Chlorophyta)

Morphological details of sexual reproduction in Carteria eugametos (Volvocales, Chlorophyta) were studied under controlled laboratory conditions. Protoplasts of the two pairing, flagellate cells were released from cell walls to become isogametes. Such gametes were nonmotile and soon fused to form a completely immobile zygote. The zygote then secreted a cell wall to enter the dormant period. After dark treatment, the zygote produced four, eight or 16 quadriflagellate germ cells, and a transparent vesicle enclosing all the germ cells was released from the zygote wall. This type of zygote germination and aplanogamy in C. eugametos is unique and may be related to its peculiar phylogenetic position within the Volvocales (Chlamydomonadales).     

The immediate induction of extensive cell fusion by Ca2+ addition in Dictyostelium discoideum

In mated cultures (NC4 X V12) of Dictyostelium discoideum containing 1.0 mM CaCl2, cell fusion generates large numbers of binucleate cells which develop into zygote giant cells. In the absence of Ca2+, binucleate formation does not occur. When 1.0 mM CaCl2 is added to Ca2+-deficient cultures at 18 h, 50% of the cells fuse within 45 min producing large multinucleate syncytia. Small, presumptive gametes appear in Ca2+-deficient cultures and reach a peak of about 20% of the cell population by 10 h, but they maintain this plateau and do not fuse. Upon the addition of CaCl2, the presumptive gametes immediately fuse, producing binucleate cells which develop rapidly into morphologically distinct giant cells. Cell fusion continues, resulting in the formation of extremely large (40-80 microns diameter) multinucleate syncytia by 45 min. The induction of this extensive, synchronous cell fusion does not occur in the presence of other chloride salts and EGTA inhibits it, revealing that Ca+ is the regulatory ion.     

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.     

Long-term production of soluble human Fas ligand through immobilization of <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis> in a fibrous bed bioreactor

The production of recombinant glycoproteins in Dictyostelium discoideum by conventional cell culture methods was limited by low cell density as well as low growth rate. In this work, cotton towel with a good adsorption capability for D. discoideum cells was used as the immobilization matrix in an external fibrous bed bioreactor (FBB) system. With batch cultures in the FBB, the concentration of immobilized cells in the cotton fiber carrier increased to 1.37 × 10⁸ cells per milliliter after 110-h cultivation, which was about tenfold higher than the maximal cell density in the conventional free-cell culture. Correspondingly, a high concentration of soluble human Fas ligand (hFasL; 173.7 μg l⁻¹) was achieved with a high productivity (23 μg l⁻¹ h⁻¹). The FBB system also maintained a high density of viable cells for hFasL production during repeated-batch cultures, achieving a productivity of 9∼10 μg l⁻¹ h⁻¹ in all three batches studied during 15 days. The repeated-batch culture using immobilized cells of D. discoideum in the FBB system thus provides a good method for long-term and high-level production of hFasL.