G protein beta subunit-null mutants are impaired in phagocytosis and chemotaxis due to inappropriate regulation of the actin cytoskeleton.

Chemotaxis and phagocytosis are basically similar in cells of the immune system and in Dictyostelium amebae. Deletion of the unique G protein beta subunit in D. discoideum impaired phagocytosis but had little effect on fluid-phase endocytosis, cytokinesis, or random motility. Constitutive expression of wild-type beta subunit restored phagocytosis and normal development. Chemoattractants released by cells or bacteria trigger typical transient actin polymerization responses in wild-type cells. In beta subunit-null cells, and in a series of beta subunit point mutants, these responses were impaired to a degree that correlated with the defect in phagocytosis. Image analysis of green fluorescent protein-actin transfected cells showed that beta subunit- null cells were defective in reshaping the actin network into a phagocytic cup, and eventually a phagosome, in response to particle attachment. Our results indicate that signaling through heterotrimeric G proteins is required for regulating the actin cytoskeleton during phagocytic uptake, as previously shown for chemotaxis. Inhibitors of phospholipase C and intracellular Ca2+ mobilization inhibited phagocytosis, suggesting the possible involvement of these effectors in the process.

RacF1, a novel member of the Rho protein family in Dictyostelium discoideum, associates transiently with cell contact areas, macropinosomes, and phagosomes.

Using a PCR approach we have isolated racF1, a novel member of the Rho family in Dictyostelium. The racF1 gene encodes a protein of 193 amino acids and is constitutively expressed throughout the Dictyostelium life cycle. Highest identity (94%) was found to a RacF2 isoform, to Dictyostelium Rac1A, Rac1B, and Rac1C (70%), and to Rac proteins of animal species (64-69%). To investigate the role of RacF1 in cytoskeleton-dependent processes, we have fused it at its amino-terminus with green fluorescent protein (GFP) and studied the dynamics of subcellular redistribution using a confocal laser scanning microscope and a double-view microscope system. GFP-RacF1 was homogeneously distributed in the cytosol and accumulated at the plasma membrane, especially at regions of transient intercellular contacts. GFP-RacF1 also localized transiently to macropinosomes and phagocytic cups and was gradually released within <1 min after formation of the endocytic vesicle or the phagosome, respectively. On stimulation with cAMP, no enrichment of GFP-RacF1 was observed in leading fronts, from which it was found to be initially excluded. Cell lines were obtained using homologous recombination that expressed a truncated racF1 gene lacking sequences encoding the carboxyl-terminal region responsible for membrane targeting. These cells displayed normal phagocytosis, endocytosis, and exocytosis rates. Our results suggest that RacF1 associates with dynamic structures that are formed during pinocytosis and phagocytosis. Although RacF1 appears not to be essential, it might act in concert and/or share functions with other members of the Rho family in the regulation of a subset of cytoskeletal rearrangements that are required for these processes.

Severe developmental defects in Dictyostelium null mutants for actin-binding proteins.

The actin cytoskeleton is implicated in many cellular processes, such as cell adhesion, locomotion, contraction and cytokinesis, which are central to any development. The extent of polymerization, cross-linking, and bundling of actin is regulated by several actin-binding proteins. Knock-out mutations in these proteins have revealed in many cases only subtle, if any, defects in development, suggesting that the actin system is redundant, with multiple proteins sharing overlapping functions. The apparent redundancy may, however, reflect limitations of available laboratory assays in assessing the developmental role of a given protein. By using a novel assay, which reproduces conditions closer to the natural ones, we have re-examined the effects of disruption of many actin-binding proteins, and show here that deletion of alpha-actinin, interaptin, synexin, 34-kDa actin-bundling protein, and gelation factor affect to varying degrees the efficiency of Dictyostelium cells to complete development and form viable spores. No phenotypic defects were found in hisactophilin or comitin null mutants.

Cell-cell signaling and adhesion in phagocytosis and early development of Dictyostelium.

Cell-cell signaling and adhesion regulate transition from the unicellular to the multicellular stage of development in the cellular slime mold Dictyostelium. Essential gene networks involved in these processes have been identified and their interplay dissected. Heterotrimeric G protein-linked signal transduction plays a key role in regulating expression of genes mediating chemotaxis or cell adhesion, as well as coordinating actin-based cell motility during phagocytosis and chemotaxis. Two classes of cell adhesion molecules, one cadherin-like and the second belonging to the IgG superfamily, contribute to the strength of adhesion in Dictyostelium aggregates. The developmental role of genes involved in motility and adhesion, and their degree of redundancy, have been re-assessed by using novel developmental assay conditions which are closer to development in nature.

Dictyostelium cytosolic fucosyltransferase synthesizes H type 1 trisaccharide in vitro.

A fucosyltransferase activity has been detected using lacto-N-biose I as acceptor in the lower eukaryote Dictyostelium discoideum. This transferase requires divalent cations and is inhibited by N-ethylmaleimide and detergent treatment. Apparent calculated Km values for GDP-Fuc and lacto-N-biose I are 1.27 microM and 2.80 mM, respectively. The activity is quantitatively recovered in the supernatant after centrifugation at 100000 x g for 1 h. The reaction product, as determined by gel permeation chromatography, sensitivity to fucosidases, and analysis of partially methylated derivatives, is Fucalpha1-2Galbeta1-3GlcNAc (H type 1 trisaccharide).

Cloning and transcriptional regulation of the gene encoding the vacuolar/H+ ATPase B subunit of Dictyostelium discoideum.

The main function of vacuolar H+ ATPases in eukaryotic cells is to generate proton and electrochemical gradients across the membrane of inner compartments. We have isolated the gene encoding the B subunit of Dictyostelium discoideum vacuolar H+ ATPase (vatB) and analyzed its transcriptional regulation. The deduced protein comprises 493 amino acids with a calculated molecular mass of 54874 Da. The predicted protein sequence is highly homologous to previously determined V/H+ ATPase B subunit sequences. The protein is encoded by a single gene in the Dictyostelium genome. The gene is maximally expressed during growth and it decreases during the first hours of development. Gene expression is rapidly enhanced by phagocytosis, but not by fluid-phase endocytosis. Acidic and alkaline conditions affect vatB gene expression differently.

Derivatization of polyacrylamide gels with pallidin, a lectin of the cellular slime mold polysphondylium pallidum.

A procedure for covalent immobilization of (glyco) proteins in polyacrylamide gels is described. Pallidin, a cytosolic and membrane protein of Polysphondylium pallidum with the properties of a lectin, was derivatized into polyacrylamide gels. Activity of the derivatized gels was assessed in a cell adhesion assay, using rabbit erythrocytes. Immobilization resulted in stabilization of the binding activity in comparison with the agglutination activity of the soluble protein.

Cell surface carbohydrates and cell recognition in Dictyostelium.

Carbohydrate ligands and complementary receptors have been detected on the surface of Dictyostelium cells, using lectins, monoclonal antibodies, and immobilized sugar probes. They have been implicated in cell recognition processes, such as phagocytosis and intercellular adhesion, and could act as membrane signals for differentiation. Specific glycoproteins have been proposed to mediate intercellular adhesion in Dictyostelium discoideum and Polysphondylium pallidum and to account for the species-specificity of adhesion displayed by these species. Recent studies with the inhibitor of N-glycosylation, tunicamycin, and with glycosylation defective mutants suggest that some carbohydrate groups in these glycoproteins play a role in cell adhesion.

Intercellular adhesion in the cellular slime mold Polysphondylium pallidum.

Evidence is reviewed involving two classes of contact sites in intercellular adhesion of the cellular slime mold P. pallidum. Characterization and purification of the contact sites by using an immunological approach have led to the identification of a dimerizing glycoprotein as the major component mediating cell adhesion. Monoclonal antibodies have confirmed its localization on the cell surface of both growth phase and aggregation competent cells. A second component, which is developmentally regulated, has been partially purified. In an independent approach, plasma membrane factors which directly affect cell adhesion have been extracted and their relationship with the immunologically defined contact sites studied. The involvement of these different adhesion factors in regulating species-specific intercellular adhesion was investigated in cell sorting out experiments. It was shown that species-specific intercellular adhesion is mediated by the developmentally regulated contact sites.

Monoclonal antibodies against Dictyostelium plasma membranes: their binding to simple sugars.

Monoclonal antibodies raised against purified membranes from Dictyostelium discoideum were classified according to three criteria: type of antigen as revealed in immunoblots, developmental regulation of the target antigens, and location of the antigens on the cell surface. Some antibodies reacted with myosin, two with glycolipids. One group of antibodies bound to the protein moiety of the contact site A glycoprotein, whereas another group reacted with carbohydrate epitopes that the contact site A glycoprotein shared with a few other membrane glycoproteins. Binding of the latter antibodies to their antigens was either specifically blocked by N-acetylglucosamine or by maltose as well as methyl-alpha-mannoside and N-acetylglucosamine. These anti-carbohydrate antibodies bound specifically to agarose beads derivatized with some sugars. These results and competition studies with several carbohydrates suggest that the epitope recognized by the antibodies contains as major components N-acetylglucosamine, maltose and alpha-mannose residues. One monoclonal antibody, which reacts with N-acetylglucosamine, was used for affinity purification of the contact site A glycoprotein from a crude membrane extract. N-acetylglucosamine was used as a mild eluent of the antigen from the antibody column. No detergents were added during the entire purification procedure.

Cell adhesion in the life cycle of Dictyostelium.

Three forms of cell adhesion determine the life cycle of Dictyostelium: i) adhesion of bacteria to the surface of the growing amoebae, as the prerequisite for phagocytosis; ii) cell-substrate adhesion, necessary for both locomotion of the amoebae and migration of the slug; iii) cell-cell adhesion, essential for transition from the unicellular to the multicellular stage. Intercellular adhesion has received the most attention, and fruitful approaches have been developed over the past 25 years to identify, purify and characterize cell adhesion molecules. The csA glycoprotein, in particular, which mediates adhesion during the aggregation stage, is one of the best defined cell adhesion molecules. The molecular components involved in phagocytosis and cell-substratum adhesion are less well understood, but the basis has been laid for a systematic investigation of both topics in the near future.

Adhesion of Dictyostelium discoideum cells to carbohydrates immobilized in polyacrylamide gels. II. Effect of D-glucoside derivatives on development.

In an accompanying report (Bozzaro, S., and Roseman, S. (1983) J. Biol. Chem. 258, 13882-13889), evidence is presented that the slime mold Dictyostelium discoideum contains three cell surface receptors specific for D-glucose, D-mannose, and N-acetyl-D-glucosamine, respectively. The synthetic probes used for these studies consisted of the sugars covalently linked to polyacrylamide gels. In the present experiments, starved cells were placed on these and other immobilized sugars to determine whether the sugar derivatives influenced normal development in this organism. When D. discoideum cells are on a solid surface under water, they form aggregation centers and strands of cells (which radiate from the center), send "signals" i.e. pulses of cyclic AMP from the center down the strands, and finally, after cells in the strands migrate to the center, form tight aggregates. These results were obtained on all polyacrylamide gel derivatives tested except one class, derivatives of D-glucose (O- and S-glucosides, cellobiosides, and maltosides). On these gels, aggregation centers and strands formed normally, but at a certain point stopped "signaling" and suddenly dissociated, with the cells rapidly migrating away from one another by negative chemotaxis (see Appendix to this report). Furthermore, a simultaneous dissociation of several centers was often observed. Following a brief period of random movement after dissociation, aggregation centers once again formed and the cycle was repeated. This cycle was repeated as often as 30 times or more over a 24-h period. The cells on the glucoside gels became aggregation-competent at the same time as the control cells, and the adhesion-dissociation cycle appeared to have no effect on the synthesis of some developmentally regulated proteins, such as UDP-glucose pyrophosphorylase. Interpretations of the phenomenon and its potential for studying gene regulation in this organism are discussed.

Adhesion of Dictyostelium discoideum cells to carbohydrates immobilized in polyacrylamide gels. I. Evidence for three sugar-specific cell surface receptors.

Dictyostelium discoideum cells appear to be able to recognize particular carbohydrate prosthetic groups at different stages in their life cycle. We therefore used our previously developed model system (consisting of polyacrylamide gels containing putative ligands covalently linked to the polymer) to determine the receptors on these cells capable of recognizing carbohydrates. D. discoideum cells, at different developmental stages from growth phase to late aggregation, were incubated with the derivatized gels, and the number of adherent cells was determined by measuring alanine transaminase after cell lysis. From 70 to 100% of the cells firmly adhered to gels derivatized with glucose, maltose, or cellobiose. The cells were also capable of binding to N-acetylglucosamine and mannose, but both the rate and the extent of binding to these sugars were less than those observed with the glucose derivatives. Furthermore, binding to N-acetylglucosamine decreased to negligible levels during the aggregation stage of development. The cells did not bind to the glucose-derivatized gels in the presence of glucose and a variety of carbohydrates containing glucose at the nonreducing termini, whereas binding was not inhibited by N-acetylglucosamine, mannose, and derivatives of these sugars. Adhesion to all sugars was blocked by 2,4-dinitrophenol. This inhibitor also reversed the binding to gels containing N-acetylglucosamine and mannose, but not to glucose. Differential binding to the three monosaccharides was also observed under conditions affecting the normal amoeboid shape of the cells. In addition, adhesion to N-acetylglucosamine and mannose was trypsin-sensitive, whereas adhesion to glucose was only slightly affected by treating the cells with trypsin (and cycloheximide). These and other results suggest that D. discoideum cell adhesion to derivatized gels is mediated by three different receptors, one highly specific for glucose and two (probably less specific) for N-acetylglucosamine and mannose.

Cyclic GMP regulation and responses of Polysphondylium violaceum to chemoattractants.

In cells of the cellular slime mold Polysphondylium violaceum an attractant, which is released during the aggregation stage, causes a transient rise of the cyclic GMP concentration. Cells of this organism develop in shaken suspensions after they have finished growth. Cell development is not accompanied by an increase in the EDTA stability of cell adhesion. Both the developmental regulation and the specificity of chemotactic responses is reflected in the light scattering patterns recorded in cell suspensions: Folic acid causes a strong response in early preaggregation cells and the Polysphondylium attractant does the same in aggregation competent cells, whereas cyclic AMP is inactive in both stages.

Monoclonal antibodies that block cell adhesion in Polysphondylium pallidum: reaction with L-fucose, a terminal sugar in cell-surface glycoproteins.

By permethylation analysis the linkages of L-fucose and D-mannose in the oligosaccharide residues of cell surface glycoproteins of Polysphondylium pallidum were determined. Mannose was found in terminal positions, in 1,2-, 1,3- and 1,6-intra-chain linkages, and at branch points. Fucose was exclusively located at nonreducing ends. Fab of a monoclonal antibody, mAb 293, has been previously shown to inhibit cell adhesion in P. pallidum completely. Binding of this antibody to glycoprotein was blocked by L-fucose, and at very high concentrations also by D-mannose. The dissociation constant for the antibody-fucose complex was Kd = 70 microM, which was two orders of magnitude higher than estimated for the natural oligosaccharide. Antibody-glycoprotein complexes dissociated in the presence of 100 mM L-fucose with a half-time of about 56 s. The blockage by L-fucose is taken as evidence that the adhesion-blocking antibody binds to oligosaccharide end groups containing L-fucose as the terminal sugar.

Regulation of gene expression in Dictyostelium discoideum cells exposed to immobilized carbohydrates.

When amoebae of Dictyostelium discoideum develop on gels of polyacrylamide that are derivatized with glucosides, they become capable of aggregation at the same time as cells not exposed to glucosides. However, the aggregation centers and streams of adherent cells formed on immobilized glucosides suddenly disintegrate. The cells repeatedly re-aggregate, but never form tight aggregates as they do on other substrata. Tight aggregates formed in the absence of glucosides disperse after their transfer to glucoside gels, and the cells undergo aggregation-disaggregation cycles. The formation of tight aggregates is correlated with the expression of specific post-aggregative poly(A) RNAs. These RNAs are not expressed in cells developing on glucoside gels, and the dispersal of tight aggregates on such gels is accompanied by the almost complete loss of these RNAs. A developmentally regulated membrane glycoprotein called contact site A, which is a marker of aggregation-competent cells, is normally expressed on glucoside gels. Cyclic AMP is also produced, indicating that the strong increase of adenylate cyclase activity during the preaggregation phase is not affected. In conclusion, cell contact with immobilized glucosides specifically inhibits postaggregative gene expression and arrests development at the aggregation stage.

Dictyostelium cells produce platelet-activating factor in response to cAMP.

Evidence is provided that Dictyostelium discoideum cells produce 1-O-alkyl-2-delta-acetyl-O-sn-glycero-3-phosphocholine (platelet-activating factor, PAF). D. discoideum PAF has been characterized as being identical with mammalian platelet-activating factor, based on its stimulation of rabbit platelet aggregation, its physicochemical properties and mass spectrum. The basal activity of PAF increases after starvation and during aggregation and declines at the slug stage. PAF is not detected in the extracellular space. Cell treatment with cAMP pulses stimulates a transient accumulation of PAF, probably via activation of a cAMP-dependent acetyltransferase, suggesting a possible involvement of PAF in cAMP-regulated processes in Dictyostelium.

Beta-1,3-galactosyltransferase and alpha-1,2-fucosyltransferase involved in the biosynthesis of type-1-chain carbohydrate antigens in human colon adenocarcinoma cell lines.

We studied the beta-1,3-galactosyltransferase (GalT) and alpha-1,2-fucosyltansferase (FT) involved in the biosynthesis of type-1-chain carbohydrate antigens in human colon adenocarcinoma cell lines. We detected a GalT activity able to use GlcNAc as acceptor and found that lacto-N-biose I (Galbeta1-3GlcNAc) is the only reaction product. Such beta1,3GalT is kinetically similar to a pig trachea enzyme involved in mucin synthesis. The specific activity is high in cells that react strongly with anti-Lewis a and anti-Lewis b antibodies, and undetectable in a cell line that lacks antibody reaction. Reverse-transcriptase-mediated PCR analysis followed by DNA sequencing indicated that secretor-type alpha1,2FT is expressed in the cells, while the H type alpha1,2FT is not. The apparent Km values for donor and acceptor substrates determined for alpha1,2FT are similar to those of secretor-type alpha1,2FT and the specific activity measured correlates with Lewis b antigen expression on the cell surface. Moreover, some of the cell lines express Lewis y and H type 2 antigens, indicating that secretor type alpha1,2FT is responsible for their synthesis. Results suggest that biosynthesis of type-1-chain tumor-associated antigens in human colon carcinoma cells is operated by secretor-type alpha1,2FT, as reported in normal mucosa, and that beta1,3GalT activity may play a relevant role in its control.

Detection of subtle phenotypes: the case of the cell adhesion molecule csA in Dictyostelium.

Dictyostelium amoebae aggregate into a multicellular organism by cAMP-driven chemotaxis and cell-cell adhesion. Cell adhesion is mediated by an EDTA-sensitive and an EDTA-resistant adhesion system. The latter is developmentally regulated and triggered by homophilic interactions of the membrane glycoprotein csA; on disruption of the encoding gene, EDTA-resistant contacts fail to form. Nevertheless, csA-null cells under usual laboratory conditions aggregate normally and complete development. By using experimental conditions that reproduce more closely the habitat of Dictyostelium amoebae, evidence is provided that csA is required for development and that its expression confers a selective advantage to populations of wild-type cells over csA-null mutants. The latter display reduced cell-cell adhesion, increased adhesiveness to the substratum, and slower motility, which lead to their sorting out from aggregating wild-type cells. It is proposed that the experimental conditions commonly used in the laboratory are not stringent enough to assess the developmental role of csA and other proteins. The assay described can be used to detect subtle phenotypes, to reexamine the developmental role of apparently nonessential genes, and to test the validity of recent models on emergence and maintenance of apparent genetic redundancy.