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

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.     

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.     

Possible existence of a light-inducible protein that inhibits sexual cell fusion in Dictyostelium discoideum.

Sexual cell fusion is an initial step of macrocyst formation in Dictyostelium discoideum and requires environmental conditions such as darkness, plenty of water and the presence of calcium ions. We have been analyzing the mechanism of sexual cell fusion between HM1 and NC4, heterothallic strains in D. discoideum. Cells of these strains have been shown to be fusion competent when cultured in a liquid medium in darkness, but not so when cultured on agar plates or in a liquid medium in the light. Two cell-surface proteins, gp70 and gp138, have been identified as target molecules for fusion-blocking antibodies and therefore as relevant to sexual cell fusion. In the present study, gp70 was shown to be present in HM1 cells cultured in the light, and fusion incompetent. Intact HM1 cells cultured in the light were unable to absorb the fusion-blocking activity of antibodies against membrane components of fusion-competent HM1 cells, whose activity had been shown to be absorbed by gp70, but they did so after separation of proteins in the SDS-PAGE. In addition, fusion-competent HM1 cells were found to lose their fusion competence by subsequent cultivation in the light. This loss of competence was cycloheximide sensitive, indicating that de novo synthesis of proteins was necessary for this inhibition. From these results, we presume that light induces a protein that hinders the interaction of gp70 in HM1 cells with its receptor on the NC4 cell surface and thereby inhibits the sexual process between these strains.     

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.     

Isolation and Characterization of Dictyostelium Mutants Defective in Sexual Cell Fusion

Sexual cell fusion in the cellular slime mold Dictyostelium discoideum occurs between cells of opposite (heterothallic system) or same (homothallic system) mating types. It also requires certain environmental conditions such as darkness and abundance of water, and thus offers an interesting model system for analyzing mechanisms of cell recognition and of cellular response to environmental factors. We have been studying the mechanism of sexual cell fusion, using two heterothallic strains, NC4 and HM1 of D. discoideum. Two cell-surface glycoproteins, gp70 and gp138, have been identified as relevant molecules in the cell fusion of these strains. The former is specific to mat a cells (HM1) and the latter, common to both mat a and mat A (NC4). Involvement of cell-surface carbohydrates has also been suggested. However, the fuctions of the above fusion-related molecules are still elusive. In the present study, we isolated fusion-deficient mutants from a mutagenized mat A strain of D. discoideum to set up combined genetic and biochemical analyses. Among the three nonconditional mutants obtained, two were normal in the fruiting-body formation, asexual development, but one was aggregateless ( agg ⁻). Further analysis of these mutants would provide detailed information on the mechanism of 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.     

Involvement of Cell Surface Carbohydrates in the Sexual Cell Fusion of Dictyostelium discoideum

In order to analyze the molecular mechanism of sexual cell fusion between cells of HM1 and NC4 (opposite mating type strains in Dictyostelium discoideum ), monoclonal antibodies were raised against partially-purified gp 70, a fusion-related protein of HM1 cells. The antibodies were screened for activity to inhibit cell fusion and 9 hybridoma clones were obtained. One of the fusion-blocking monoclonal antibodies, mAb1G7, was used for further analysis. It recognized nearly ten bands in an immunoblot of fusion competent HM1 cells, but no bands when HM1 membrane proteins had been deglycosylated. These results suggest the importance of carbohydrates in the cell fusion process. To confirm this possibility, effects of sugars or lectins on cell fusion were examined. Although inhibition by the sugars was incomplete, Con A, WGA, LCA, strongly inhibited cell fusion. Furthermore, tunicamycin inhibited the acquisition of fusion competence in HM1 cells, indicating the importance of N-linked glycosylation of proteins in cell fusion. All above results suggest that N-linked carbohydrates on HM1 cell surface are involved in the sexual cell fusion of D. discoideum .     

A new member of the GP138 multigene family implicated in cell interactions in Dictyostelium discoideum.

The cellular slime mold Dictyostelium discoideum reproduces sexually under submerged and dark conditions. Its mating system is polymorphic and particularly interesting with respect to mechanisms of cell recognition. The cell-surface glycoprotein gp138 has been implicated in sexual cell interactions, as it was identified as a target molecule for the antibodies that block sexual cell fusion in D. discoideum. Two mutually homologous genes, GP138A and GP138B, have been cloned, but gene disruption experiments to clarify their functional relationships suggested that there is at least one more gene for gp138. Further protein analysis including peptide mapping also revealed that gp138 exists as three isoforms, DdFRP1, DdFRP2, and DdFRP3. GP138A encodes DdFRP2 and GP138B, DdFRP3, and the presence of a third gp138 gene encoding DdFRP1 was suggested. Here, we isolated and characterized a third GP138 gene, GP138C. Although the deduced amino acid sequences of GP138C matched completely with those of peptide fragments of DdFRP1 in the N-terminal half, the rest did not give complete matches. Overexpression of GP138C caused an increase in the intensity of DdFRP1, but disruption of this gene did not diminish DdFRP1. Our results indicate that GP138C encodes a protein very similar to but distinct from DdFRP1. The GP138 multigene family is thus composed of more members than previously expected, and their functional relationships are of special interest.     

Targeted disruption of genes for gp138, a cell-fusion-related protein in Dictyostelium discoideum, revealed the existence of a third gene

The sexual cycle of the cellular slime mold, Dictyostelium discoideum , offers a suitable experimental system to analyze sexual cell interactions. We have been analyzing molecular mechanisms involved in sexual cell fusion using complementary heterothallic strains in D. discoideum and have identified several cell surface proteins involved in the process. One of them, gp138 is present in strains of both mating types and considered to be responsible for membrane fusion itself. Two genes with high mutual homology, GP 138 A and GP 138 B , have been identified so far as encoding this protein. Expression of antisense RNA for GP 138 B has been shown to suppress sexual cell fusion, confirming the critical importance of these genes in sexual cell fusion. However, neither the functional relationship of the two gp138 genes nor the possibility of the existence of more genes that encode gp138 has been determined yet. In the present study, GP 138 A and GP 138 B were disrupted by homologous recombination in an effort to clarify these points. Analysis of the double knock-out mutants suggested the presence of a third gene for gp138.     

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.     

Self- and non-self-recognition in bisexual mating of Dictyostelium discoideum

Cell recognition plays a central part in the sexual process. Although cell-surface molecules involved in gamete recognition have been identified in several organisms, our knowledge of the molecular basis of sexual cell recognition is still limited. We have been studying molecular mechanisms of sexual cell fusion using the lower eukaryote Dictyostelium discoideum . There are homothallic, heterothallic, bisexual and asexual strains in D. discoideum , and how they distinguish between each other to find out proper partners is an interesting and important question. However, analytical studies of sexuality in D. discoideum have been carried out mostly on heterothallic strains, and the polymorphism of the mating system has not yet been thoroughly investigated. In the present study, we extended our analysis to the bisexual mating phenomenon paying special attention to the mechanism of self-incompatibility. We showed that a bisexual strain WS2162 was self-incompatible at the step of sexual cell fusion. Results of antibody inhibition of cell fusion and detection of gp138, a cell-fusion-related protein found in heterothallic strains, suggest that a molecular basis for bisexual and heterothallic mating are common. We propose two models to clarify the mechanisms of self- and non-self discrimination in bisexual mating patterns of D. discoideum .     

Concanavalin A and wheat germ agglutinin binding glycoproteins associated with cell fusion and zygote differentiation in Dictyostelium discoideum: effects of calcium ions and tunicamycin on glycoprotein profiles

To determine which glycoproteins may be critical to sexual development in Dictyostelium discoideum, cell samples from different developmental stages were separated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis and blotted to nitrocellulose. Concanavalin A (ConA) and wheat germ agglutinin (WGA) binding proteins were visualized on the blots using an immunochemical procedure employing peroxidase-antiperoxidase. ConA labelled at least 28 proteins, but only one band showed calcium-dependent changes in its expression. WGA bound at least 30 proteins and changes in several bands were observed that did not occur in calcium-deficient controls. Two WGA-binding glycoproteins which migrated at 200 and 166 kilodaltons (kDa), respectively, showed developmental changes associated with the time of cell fusion. One WGA-binding and one ConA-binding glycoprotein migrating at 130 and 126 kDa, respectively, appeared later during sexual development, in association with the phase of zygote differentiation. Several WGA- and ConA-binding glycoproteins decreased during sexual development, but were not affected by the absence of calcium ions. Tunicamycin (1 microgram/mL) inhibited cell fusion when added to sexual cultures prior to the appearance of the 166-kDa glycoprotein gp166. The effects of this inhibitor on development support the importance of glycoproteins to cell fusion during sexual development in 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.     

Total tetra knockout of GP138 multigene family implicated in cell interactions in Dictyostelium discoideum.

The cellular slime mold Dictyostelium discoideum reproduces sexually under submerged and dark conditions. A cell surface glycoprotein gp138 has been identified as a target molecule for cell fusion-blocking antibodies, and is considered to be indispensable for the sexual cell fusion in this organism. Currently, four isoforms of gp138, DdFRP1alpha, DdFRP1beta, DdFRP2, and DdFRP3, are known. Genes encoding the latter three isoforms, GP138C, GP138A, and GP138B, have been isolated, comprising a GP138 multigene family. Here we isolated the fourth GP138 gene, GP138D, encoding DdFRP1alpha. These GP138 genes were found to cluster in a tandem array on chromosome 5, being bordered by two GP138-like sequences highly homologous to them but truncated. To clarify functional relationships among the GP138 family members, the entire GP138 region was deleted by a single knockout. Northern hybridization and western immuno-blotting analyses confirmed complete losses of GP138 mRNA and DdFRPs in the knockout strains, indicating that there are no more GP138 genes. Unexpectedly, however, the GP138-null mutants were fully potent for both sexual cell fusion and subsequent development. In addition, the original fusion-blocking antibodies detected a cell surface protein of close electrophoretic mobility to gp138 in the knockouts, suggesting the possibility that the actual target molecule of the fusion-blocking antibodies was not DdFRPs but this unidentified component. Since GP138-null mutants exhibited no obvious defects either in growth or asexual development, the real function of the GP138 family is unknown. Nevertheless, the expression levels of other developmental genes such as acaA, csaA, cotA-C, and spiA appeared to be altered in the GP138-null mutants. Therefore, it seems to have a non-critical but some role(s) during asexual development.     

An analysis of discoidin I binding sites in Dictyostelium discoideum (NC4).

Vegetative wild-type (strain NC4) D. discoideum cells and cells at the 10h stage of development (aggregation) were harvested in the presence of 0.5 M-galactose to remove any endogenous discoidin I already bound to the cell surface, and fixed with glutaraldehyde. Affinity-purified 125I-labelled discoidin I bound to these fixed cells in a specific manner, greater than or equal to 95% of binding being inhibited by 0.5 M-galactose. Binding of 125I-labelled discoidin I was essentially complete in 90 min at 22 degrees C. Based on specific radioactivity measurements, vegetative (0h) D. discoideum (NC4) cells bind approx. 8.4 x 10(5) discoidin I tetramers/cell and aggregated (10h) cells bind 5.1 x 10(5) discoidin I tetramers/cell, each exhibiting apparent positive co-operativity of binding with highest limiting affinity constants (Ka) of approx. 1 x 10(7) and 2 x 10(7) M-1, respectively. Klebsiella aerogenes, the food source used for growth of D. discoideum NC4 amoebae, also binds 125I-labelled discoidin I and this is greater than 99% inhibited by 0.5 M-galactose. However, at the levels of bacterial contamination present, greater than 97% of 125I-labelled discoidin I binding to D. discoideum cell preparations was to the cells themselves. Confirmation of the number of discoidin I tetramers bound per D. discoideum cell was obtained by elution of bound 125I-labelled discoidin I followed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and then quantification by scanning of stained discoidin I bands.     

Common Cell-Surface Receptors for Discoidin I and Discoidin II in Dictyostelium discoideum

Following nutrient depletion, cells of the cellular slime mould Dictyostelium discoideum become cohesive and aggregate to form multicellular complexes. Several proteins that accumulate on the cell surface during this period have been implicated in mediating aggregative-phase cell cohesion, namely contact sites A (CsA), gp 150, and two endogenous lectins (discoidin I and discoidin II). The aggregating cells also possess receptors for both discoidin I and discoidin II but these have not yet been isolated and characterised for both lectins.
In the present study we investigated the relationship between the receptors for these lectins, in particular to what extent discoidin I and discoidin II receptors are common. Radio-iodinated discoidin I and discoidin II were purified and used in binding assays for lectin receptors on the surface of aggregated (10 h stage of development) D. discoideum NC4 cells. Sugar competition of ¹²⁵I-labelled discoidin I and ¹²⁵I-labelled discoidin II binding indicated distinct but overlapping sugar specificities for these lectins when binding to their in vivo receptors. Competition of the binding of radio-iodinated lectin with either unlabelled discoidin I or unlabelled discoidin II showed that at least 50% of the cell-surface binding sites for these lectins are in common and for these receptors the binding affinity of discoidin I is 9–20 times higher than for discoidin II. Approximately 35% of discoidin II binding sites appear to be unavailable for discoidin I binding.     

Molecular and biological characterization of fusion regulatory proteins (FRPs): anti-FRP mAbs induced HIV-mediated cell fusion via an integrin system.

Anti-FRP mAbs induced polykaryocyte formation of U2ME-7 cells (CD4+U937 cells transfected with the HIV gp160 gene). Anti-FRP-1 mAb immunoprecipitated gp80-85, gp120 and homodimers of these peptides, and anti-FRP-2 mAb reacted with gp135 identically to the alpha 3 subunit of integrin. Both anti-FRP-1 and anti-FRP-2 mAb-induced cell fusion was blocked by anti-beta 1 integrin antibody, fibronectin or inhibiting anti-FRP-1 antibody. Therefore, anti-FRP mAbs were thought to induce the fusion via an integrin system(s). FRP-mediated fusion was temperature, cytoskeleton, energy and Ca2+ dependent. These experiments showed a possible regulatory function of cell fusion by an integrin system(s).     

Trypsin-sensitive and -resistant components in human T-cell membranes required for syncytium formation by human T-cell lymphotropic virus type 1-bearing cells.

Human T-cell lymphotropic virus type 1 (HTLV-1) envelope proteins play an important role in viral entry into target cells. In a syncytium formation assay consisting of a coculture of HTLV-1-bearing cells and target cells, mature gp46 and gp21 proteins each inhibited syncytium formation induced by HTLV-1-bearing cells. Experiments with 125I-labeled proteins showed that 125I-gp46 bound specifically with MOLT-4 target cells even in the presence of large amounts of gp21, whereas 125I-gp21 binding to target cells was completely blocked in the presence of large amounts of gp46. These observations suggest that HTLV-1 envelope proteins in syncytium formation interact with at least two components, which are located close to each other on the cell membrane. We isolated two components from MOLT-4 cell lysate, using Sepharose 4B columns coupled with peptides corresponding to amino acids 197 to 216 and 400 to 429, respectively, of the envelope protein. One is a trypsin digestion-sensitive component of approximately 34 to 35 kDa, which interacts specifically with gp46. The other is a nonprotein component, which interacts with gp21. This component was destroyed by sodium periodate oxidation and was partitioned into the methanol-chloroform phase. These observations suggest that these two components play an important role in HTLV-1 entry into target cells via membrane fusion.     

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.