Ammonia Determines the Alternative Pathways of Sexual or Asexual Development in the Cellular Slime Mold Dictyostelium discoideum

The sexual development, macrocyst formation, of Dictyostelium discoideum is initiated by sexual fusion of cells. The sexual fusion is only taken place under the culture conditions of excess water and darkness. Under these conditions, cells acquire the fusion competence, but lose it when cell density is high. The loss of the fusion competence is caused by accumulation of ammonia excreted by cells in a culture. Ammonia suppresses the fusion competence of cells at a certain concentration, and consequently inhibits formation of macrocysts and induces fruiting-body formation. Thus, excess water induces the sexual development by diluting ammonia and lack of water induces the asexual development.     

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.     

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.     

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.     

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.     

Reverse genetic analyses of gamete-enriched genes revealed a novel regulator of the cAMP signaling pathway in Dictyostelium discoideum

Sexual development in Dictyostelium discoideum is initiated by the fusion of opposite mating type cells to form zygote giant cells, which subsequently gather and phagocytose surrounding cells for nutrition to form macrocysts. Here we performed the targeting of 24 highly gamete-enriched genes we previously isolated, and successfully generated knockout mutants for 16 genes and RNAi mutants for 20 genes including 6 genes without disruptants. In the knockout mutants of two genes, cell aggregation toward the giant cells was much less extensive and many cells remained around poorly formed macrocysts. We named these genes tmcB and tmcC. Although macrocyst formation of wild type cells was suppressed by the addition of exogenous cAMP, that of knockout mutants of tmcB was much less sensitive. The mRNA level of phosphodiesterase (pde) was higher and that of its inhibitor (pdi) was lower in the latter cells compared to the parental strains during sexual development. Thus, tmcB appeared to be a novel regulator of the cAMP signaling pathway specific to sexual development. Knockout mutants of tmcC were indistinguishable from the wild type cells with respect to the cAMP response, suggesting that this gene is relevant to other processes.     

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.     

Nuclear Behavior in Artificially Induced Multi-Nuclear Cells of Dictyostelium discoideum

The culture medium of the strain CK-8 of the cellular slime mold Polysphondylium pallidum contains a cell-fusion induction factor. Cells of the two opposite mating type strains NC-4 and HM1 of Dictyostelium discoideum were treated to induce cell fusion with the diluted fraction of CK-8 cultures, F2, which contains the factor and consequently numerous multinuclear cells were produced. NC-4 and HM1 usually fuse in the sexual cycle and form large multinuclear cells, called giant cells, which develop into macrocysts. These cells are very similar in morphology to the multinuclear cells produced following F2 treatment, however, the latter cells did not develop into macrocysts. In the sexually formed multinuclear cells, only two haploid nuclei fused to form a diploid nucleus and all others degenerate as previously reported. However, in the artificially produced multinuclear cells, no nuclear-fusion and degeneration took place. They stayed as heterokaryons and seem to lyse within 20 h incubation.     

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.     

Protein Synthesis Initiated by Cell Fusion in Dictyostelium discoideum

D. discoideum has two alternative developmental pathways. If cells of two complement mating-type strains, NC4 and HM1, fuse sexually, a giant cell is produced which subsequently develops into a macrocyst, the sexual structure of this organism. However, if fusion fails to occur and cells are starved, a fruiting-body is produced instead of a macrocyst. In this paper, a two-dimensional polypeptide gel electrophoresis study showed that giant cells produce specific polypeptides which may possibly be involved in macrocyst development. Out of total 497 polypeptides which appeared in a giant cell during an incubation period of 13 hr, 92 were the specific for giant cells. Four of these polypeptides were appeared within only 1 hr after the cell fusion. The other 405 were non-specific polypeptides which appeared in both giant cells and NC4 or/and HM1 cells. However, the patterns and the rates of production of each polypeptide during the incubation period were different between these cells.     

Induced cell fusion inForaminifera

Summary The fusion of cells of the monothalamous foraminiferMyxotheca arenilega (Protozoa, Rhizopoda) is described. The fusion process is induced by the separation of the extracellular organic shell from the cell body. As several cells can fuse either stepwise or simultanously, a giant cell of some millimeters in diameter can be formed.     

Breakdown of self/nonself recognition in cannibalistic strains of the predatory slime mold, Dictyostelium caveatum

Dictyostelium caveatum amebas feed upon both bacteria and the amebas of other cellular slime molds. The capacity to feed extensively upon other cellular slime molds is unique to D. caveatum amebas. They are able to phagocytose amebas larger than themselves by nibbling pieces of the cells until they are small enough to ingest. Here we report the isolation from previously cloned stock cultures of stable, cannibalistic strains of D. caveatum in which self/nonself recognition has broken down. Because of the extensive cannibalism, amebas of these strains do not complete multicellular development, and instead wander about for long periods while feeding upon each other. Although the cannibalistic behavior resembles that exhibited by the presumably diploid giant cells in the sexual cycle of other cellular slime molds, these strains are haploid and do not form macrocysts.     

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.     

An auto-inhibitor of zygote giant cell formation in Dictyostelium discoideum

The first observable event of sexual development in mated cultures (NC4x V12) of Dictyostelium discoideum is the appearance of zygote giant cells which are the product of cell fusions. The increase in the number of giant cells, which begins at about 21 h, is complete by 28 h. In this paper we show that a low molecular weight auto-inhibitor of zygote giant cell formation is present in 28-h cultures. The data presented suggest that this auto-inhibitor is a natural regulator of sexual development in D. discoideum.     

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.     

Lectin binding and inhibition studies reveal the importance of D-glucose, D-mannose and N-acetylglucosamine during early sexual development of Dictyostelium discoideum

Fluorescein-conjugated and non-conjugated lectins were used to determine which surface sugars are involved in the early events of sexual (macrocyst) development in Dictyostelium discoideum. Only zygote giant cells showed unique binding of FITC-WGA and FITC-PNA while all cell types (amoebae, gametes, binucleates, giant cells) showed identical patterns of FITC-Con A, -Gorse and -RCA II binding. In spite of its non-selective labelling of all cell types, Con A inhibited macrocyst formation. The temporal addition of Con A with and without specific hapten sugars indicates the importance of both D-mannose and D-glucose in phagocytosis and, possibly, cell fusion. WGA also inhibited macrocyst formation. Varying the time of addition of the lectin plus/minus its primary hapten sugar implicates N-acetylglucosamine as being important in cell fusion. Neither Gorse, RCA II nor PNA had any detectable inhibitory effects on macrocyst development leaving the appearance of increased PNA receptors at the giant cell surface as an enigma.     

夜光藻有性繁殖研究进展

夜光藻是全球最主要的赤潮生物之一,也是我国近海常见的浮游甲藻。根据营养方式分为异养的红色夜光藻和混合营养的绿色夜光藻,前者广泛分布于温带和亚热带近岸水域,后者仅分布于热带西太平洋、阿拉伯海、阿曼湾和红海。夜光藻的生活史包括无性繁殖和有性繁殖过程。少部分营养细胞自发转变为配子母细胞,启动了有性繁殖。每个配子母细胞可形成大量配子,具有横沟、纵沟和2根鞭毛,形态与裸甲藻接近。配子两两融合形成合子,合子不经过休眠孢囊阶段直接发育成新的营养细胞。目前,对配子母细胞形成的调控机制、合子发育的影响因素的认识还存在分歧。研究发现,营养细胞经过一定次数的二分裂后都会转变为配子母细胞,而配子的存在能够中止此过程,使营养细胞继续进行二分裂。因此,有性繁殖可能通过产生新个体对种群增长做出贡献,还可能通过释放配子维持无性繁殖,进而促进种群增长。配子在相模湾水域全年都有分布,其丰度峰值与营养细胞丰度峰值同步或提前出现,配子的大量出现可能是赤潮形成的必要条件。对有性繁殖的研究佐证了夜光藻在甲藻的系统进化中处于较为古老的地位。此外,还简单介绍了研究夜光藻有性繁殖的主要方法,回顾了国内的夜光藻研究,并对相关研究进行了展望。     

The developmental capacity of various stages of a macrocyst-forming strain of the cellular slime mold, Dictyostelium mucoroides.

Vegetative cells of certain strains of Dictyostelium mucoroides form fruiting bodies on an agar surface and macrocysts when placed under saline. This study sought to determine whether the aggregation and pseudoplasmodial stages of fruiting body formation could be induced to form macrocysts when placed under saline. Likewise, different stages in macrocyst formation were put on an agar surface to determine their potential to switch to fruiting body formation. It was found that 78% of the aggregates and 21% of the pseudoplasmodia placed under saline formed macrocysts indicating that as fruiting body development proceeds, there is a restriction of the capability of cells to respond to environmental conditions favoring macrocyst formation. Stages in macrocyst development prior to the formation of precysts always formed fruiting bodies when put on agar. Once precysts had formed, surrounded by their acellular sheath, they always developed as macrocysts on agar. Peripheral cells isolated from precysts and put on agar quickly aggregated; the aggregates became surounded by a sheath and developed as macrocysts. If isolated peripheral cells were allowed to proliferate on the agar surface, the resulting cells aggregated and formed fruiting bodies.     

Promotion of Zygote Formation by Protein Kinase Inhibitors during the Sexual Development of Dictyostelium mucoroides

To analyze the possible involvement of protein kinases in the sexual development (macrocyst formation) of the cellular slime mold Dictyostelium mucoroides-7 (Dm7), the effects of several protein kinase inhibitors were examined. K252a, a potent inhibitor of protein kinase activities, promoted the sexual cell fusion, through enhancement of gamete formation. In contrast, staurosporine (structurally and functionally similar to K252a) inhibited markedly the progress of development including cell aggregation, thus resulting in the failure of cells to form mature macrocysts. The effective period of K252a was 5–7 hr after starvation, during which Dm7 cells could acquire fusion competence, and the inhibitory effect of cAMP on zygote formation was nullified by the co-application of K252a. Although KT5720 (a specific inhibitor of cAMP-dependent protein kinase) and W7 (a calmodulin inhibitor) had no effects on zygote formation when applied separately, their combined application enhanced zygote formation like K252a did. Neither calphostin C (a specific inhibitor of Ca²⁺-dependent protein kinase) nor herbimycin A (a specific inhibitor of tyrosine kinase) exerted a stimulative influence upon macrocyst formation. These results strongly suggest that the two signal transduction pathways mediated by cAMP-dependent protein kinase (PKA) and calmodulin are closely related to zygote formation, their blockage being favorable to zygote formation.     

The ultrastructure of the eyes of<Emphasis Type="Italic"> Paravortex karlingi</Emphasis> (Plathelminthes,Rhabdocoela)

Both eyes of Paravortex karlingi belong to the rhabdomeric type. Each eye consists of a single pigment cup cell and three sensory cells. Modified mitochondria lie in three protrusions of the cup cell, and several of these mitochondria form giant mitochondrial derivates. The centres of the derivates include electron-dense substances. These modified structures may have lenticular functions. Furthermore, it is shown that giant mitochondrial derivates develop by fusion of smaller ones. Embryos have many small mitochondria in the developing eyes whereas the adult possesses less numerous, but giant forms. This circumstance leads to the assumption that all such lenticular structures within the rhabdocoels result from identical processes.