Negative chemotaxis in cellular slime molds.

This study confirms the suggestion of earlier workers that the vegetative amoebae of Dictyostelium repel each other while those of Polysphondylium violaceum do not. When Dictyostelium amoebae were placed in drops on thin and thick agar, the cells moved out faster on the thin agar, presumably because the repellent was more concentrated. This did not occur with Polysphondylium amoebae. Also, if 2 drops of cells were placed side by side, or a single drop was placed near an edge, in Dictyostelium there were fewer cells emerging between the drops (or near an edge) than on the far side. Polysphondylium showed no such difference. However, Polysphondylium amoebae were repelled by Dictyostelium cells (but not vice versa) when drops of each were placed beside one another. Finally, if Dictyostelium discoideum cells were placed in drops over thick and thin agar, but separated from the agar by a dialysis membrane, the cells again spread farther on the thin agar, indicating that the repellent is a dialyzable molecule.     

The 3'-noncoding region of the chick myosin light-chain gene hybridizes to a family of repetitive sequences in the slime mold Dictyostelium discoideum

During studies aimed at isolating myosin-specific genomic clones in Dictyostelium, we probed a lambda genomic library with a chicken myosin light-chain sequence (pML10). Many lambda recombinant Dictyostelium clones hybridized to the pML10 cDNA insert, indicating that this sequence was reiterated in the Dictyostelium genome. It was found that the 3'-noncoding region (pML10-NC) alone was responsible for these results. Dictyostelium DNA contained approximately 65 copies of a sequence(s) similar but not identical to that of pML10-NC. Southern blot analysis showed that pML10-NC hybridized to many Dictyostelium genomic DNA fragments of varying sizes generated by digestion with EcoRI, HindIII, or AluI. In addition, each of the Dictyostelium clones was different in its size, restriction map, and flanking sequences. It seems likely, therefore, that the sequences which hybridized to pML10-NC are scattered throughout the Dictyostelium genome and similar but not identical to each other or to pML10-NC. Thus, probing with pML10-NC has allowed us to select a family of closely related but not identical sequences. These D. discoideum sequences are not found in other slime mold species. No RNA complementary to pML10-NC was found in vegetative cells, 18 h culmination stage, spores, or 1- and 2-h germinating spores. pML10-NC-related sequences were present in two other Dictyostelium species but were absent in the related genus Polysphondylium.     

Developmentally regulated cell-cell adhesion in Dictyostelium purpureum is mediated by a glycoprotein synthesized in nonadhesive cells

Upon starvation the cellular slime mold, Dictyostelium purpureum, develops a form of cell-cell adhesion aiding in the formation of large multicellular aggregates, which are capable of further differentiation. The molecule that mediates this adhesion is a glycoprotein of Mr approximately 40,000. The protein shares a common carbohydrate epitope with another well-characterized cell adhesion molecule from Dictyostelium discoideum, contact sites A, but the polypeptides to which it is attached differ for each species. Although mediating a developmental form of adhesiveness, the protein is synthesized in vegetative cells at a time when they do not adhere. Most of the vegetative protein is associated with cell membranes and appears to be on the surface of these cells. The protein is compared to other cell adhesion molecules from other species of cellular slime molds, and possible explanations for its inability to function in vegetative cells are discussed.     

Nuclear ribonucleoprotein particles in the cellular slime mold Dictyostelium discoideum

The nuclear ribonucleoprotein (RNP) particles containing rapidly labeled RNA were isolated from interphase cells of the cellular slime mold Dictyostelium discoideum and characterized. The size of the isolated RNP particles was small (10S to 50S) in comparison with that of nuclear RNP particles found in higher eukaryotes. These small RNP particles do not seem to be artifacts due to degradation during the preparation of nuclear extracts. The rapidly labeled RNA of the nuclear RNP particles was heterogeneous in size and a considerable amount contained polyadenylic acid sequences. Synthesis of RNA in the nuclear RNP particles was resistant to a relatively high concentration of actinomycin D. The protein component of the RNP particle consists of at least four proteins with molecular weights of 80,000, 66,000, 60,000, and 42,000. Thus it is suggested that almost all of the nuclear RNP particles containing rapidly labeled RNA in interphase cells are RNP complexes consisting of Heterogeneous nuclear RNA and several protein species.     

Comparison of developmentally regulated lectins from three species of cellular slime mold

Extracts of the cohesive forms of the cellular slime molds Dictyostelium discoideum, Dictyostelium mucoroides and Dictyostelium purpureum contain lectin activity, assayed as hemagglutination activity. The lectin activity from each species binds quantitatively to Sepharose 4B and can be eluted with d-galactose. The resultant purified lectins are abundant proteins representing, in the case of D. purpureum, up to 5% of the total soluble protein of cohesive cells. The preparations from each species are similar but distinct in amino acid composition and other properties. Each purified preparation gives rise to two protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the major band representing as little at 77% (D. purpureum) and as much as 96% (D. mucoroides) of the total protein in the two bands. The molecular weights of the pair of bands were different for each species, ranging between about 23 000 and 26 000. The two bands are believed to represent subunits of lectins made up of either one or a combination of these two proteins. The apparent molecular weights of the purified lectin activities determined by sucrose density gradient centrifugation were all in the range of 100 000. $\text{N-}\text{Acetyl-}\text{d}\text{-galactosamine}$ was a potent inhibitor of the hemagglutination activity of each preparation; but there were some differences in the relative inhibitory potency of a number of other saccharides. Antiserum raised against each preparation, as well as univalent antibody fragments derived from these antisera, reacted best with the antigens to which they were raised; but showed some cross reaction measured both by precipitin reactions and by inhibition of hemagglutination activity of the purified lectins. The differences between the lectins from the different species could be trivial; but they also could be important for defining specific properties of these three species which reliably segregate into colonies of a single species when grown in mixed culture.     

Function of the Dictyostelium discoideum Atg1 kinase during autophagy and development

When starved, the amoebae of Dictyostelium discoideum initiate a developmental process that results in the formation of fruiting bodies in which stalks support balls of spores. The nutrients and energy necessary for development are provided by autophagy. Atg1 is a protein kinase that regulates the induction of autophagy in the budding yeast Saccharomyces cerevisiae. In addition to a conserved kinase domain, Dictyostelium Atg1 has a C-terminal region that has significant homology to the Caenorhabditis elegans and mammalian Atg1 homologues but not to the budding yeast Atg1. We investigated the function of the kinase and conserved C-terminal domains of D. discoideum Atg1 (DdAtg1) and showed that these domains are essential for autophagy and development. Kinase-negative DdAtg1 acts in a dominant-negative fashion, resulting in a mutant phenotype when expressed in the wild-type cells. Green fluorescent protein-tagged kinase-negative DdAtg1 colocalizes with red fluorescent protein (RFP)-tagged DdAtg8, a marker of preautophagosomal structures and autophagosomes. The conserved C-terminal region is essential for localization of kinase-negative DdAtg1 to autophagosomes labeled with RFP-tagged Dictyostelium Atg8. The dominant-negative effect of the kinase-defective mutant also depends on the C-terminal domain. In cells expressing dominant-negative DdAtg1, autophagosomes are formed and accumulate but seem not to be functional. By using a temperature-sensitive DdAtg1, we showed that DdAtg1 is required throughout development; development halts when the cells are shifted to the restrictive temperature, but resumes when cells are returned to the permissive temperature.     

An anticarbohydrate monoclonal antibody inhibits cell-cell adhesion in many species of Dictyostelium but not of Polysphondylium.

The anticarbohydrate monoclonal antibody d-41 inhibits the adhesion of aggregating cells, as measured by an in vitro assay, in every species of Dictyostelium tested but in none of the species from the genus Polysphondylium. Although d-41 binds significantly to the surface of cells from both genera, the ability to inhibit adhesion correlates with the binding of the antibody to a few, mostly developmentally regulated, membrane-associated proteins in each of the species affected. Previous work in D. discoideum and D. purpureum have shown that the major d-41-b binding proteins from these species at this time in development are directly involved in the adhesion process. Therefore, the presence of the epitope on these proteins in the other species of Dictyostelium implicates them in the adhesion mechanism. The function of the carbohydrates containing the epitope is yet to be determined.     

Actin filaments undergo limited subunit exchange in physiological salt conditions

The exchange of actin filament subunits for unpolymerized actin or for subunits in other filaments has been quantitated by three experimental techniques: fluorescence energy transfer, incorporation of 35S-labeled actin monomers into unlabeled actin filaments, and exchange of [14C]ATP with filament-bound ADP. In the fluorescence energy transfer experiments, actin labeled with 5-(iodoacetamidoethyl)aminonaphthalene- 1-sulfonic acid (IAENS) served as the fluorescent energy donor, and actin labeled with either fluorescein-5-isothiocyanate (FITC) or fluorescein-5-maleimide (FM) served as the energy acceptor. Fluorescent- labeled actins from Dictyostelium amoebae and rabbit skeletal muscle were very similar to their unlabeled counterparts with respect to critical actin concentration for filament assembly, assembly rate, ATP hydrolysis upon assembly, and steady-state ATPase. As evidenced by two different types of fluorescence energy transfer experiments, less than 5% of the actin filament subunits exchanged under a variety of buffer conditions at actin concentrations greater than 0.5 mg/ml. At all actin concentrations limited exchange to a plateau level occurred with a half- time of about 20 min. Nearly identical results were obtained when exchange was quantitated by incorporation of 35S-labeled Dictyostelium actin monomers into unlabeled muscle actin or Dictyostelium actin filaments. Furthermore, the proportion of filament-bound ADP which exchanged with [14C]-ATP was nearly the same as actin subunit exchange measured by fluorescence energy transfer and 35S-labeled actin incorporation. These experiments demonstrate that under approximately physiologic ionic conditions only a small percentage of subunits in highly purified skeletal muscle or Dictyostelium F-actin participate in exchange.     

Characterization of genes which are deactivated upon the onset of development in Dictyostelium discoideum

We have identified and begun characterizations of the differential expression of 15 genes whose corresponding mRNA levels decrease during the preaggregative period of the developmental program of Dictyostelium discoideum. Upon the onset of development, the mRNAs decrease from 5- to 1000-fold over the first 8 hr. The rates of loss of each mRNA were similar to one another but distinct, and the decreases were dependent on progress through the developmental program. One exception to this dependency was observed, and the decrease in this mRNA was dependent on the absolute time after initiation of development instead of progress through development. With two exceptions, the decreases in mRNA levels were dependent on developmental conditions and were not seen when cells were shaken in starvation buffer. When the polysomal distributions of each species were examined, three classes were found: most showed no significant shifts off of polysomes upon initiation of development, two were characterized by a 20% shift to nonpolysomal RNA fractions upon development, and two gave a 40-50% shift. Collectively, these characterizations reveal differences in behavior which suggest that deactivation of genes upon initiation of development in Dictyostelium involves more than one regulatory pathway.     

Intracellular calcium during chemotaxis of Dictyostelium discoideum: a new fura-2 derivative avoids sequestration of the indicator and allows long-term calcium measurements.

During stimulation of Dictyostelium discoideum amoebae with the chemoattractant cAMP, extracellular calcium is taken up by the cells. The aim of this study was to determine the cytosolic free calcium concentration ([Ca++]i) during chemotaxis of Dictyostelium cells. In contrast to most vertebrate cells, three major drawbacks were encountered: 1) the indicator fura-2 could not be introduced into the cells by incubation with the ester form, 2) once loaded, the dye was rapidly sequestered into vesicles, 3) the organic anion transport blocker probenecid was not suitable to block sequestration. These problems were met by introducing the indicator into the cells with the scrape-loading technique adapted for use with Dictyostelium and the construction of a new fura-2 derivative, fura-2-dextran. Scrape-loading of Dictyostelium yielded up to 40% of labeled, vital cells. Fura-2-dextran fulfilled the following criteria: 1) it remained homogeneously distributed in the cytoplasm of motile Dictyostelium cells, 2) it retained the fluorescence intensity of fura-2 and the affinity for calcium binding, 3) it was very well suitable to demonstrate changes of [Ca++]i in serum-stimulated fibroblasts. [Ca++]i-measurements with fura-2-dextran in chemotactically active D. discoideum amoebae revealed that the large decrease in the extracellular calcium concentration is not accompanied by an overall change in [Ca++]i. Chemotaxis in this organism occurs in the absence of global changes in [Ca++]i. However, we cannot exclude either short-lived or local changes just beneath the plasma membrane.     

Exploitation of other social amoebae by Dictyostelium caveatum

Dictyostelium amoebae faced with starvation trigger a developmental program during which many cells aggregate and form fruiting bodies that consist of a ball of spores held aloft by a thin stalk. This developmental strategy is open to several forms of exploitation, including the remarkable case of Dictyostelium caveatum, which, even when it constitutes 1/10(3) of the cells in an aggregate, can inhibit the development of the host and eventually devour it. We show that it accomplishes this feat by inhibiting a region of cells, called the tip, which organizes the development of the aggregate into a fruiting body. We use live-cell microscopy to define the D. caveatum developmental cycle and to show that D. caveatum amoebae have the capacity to ingest amoebae of other Dictyostelid species, but do not attack each other. The block in development induced by D. caveatum does not affect the expression of specific markers of prespore cell or prestalk cell differentiation, but does stop the coordinated cell movement leading to tip formation. The inhibition mechanism involves the constitutive secretion of a small molecule by D. caveatum and is reversible. Four Dictyostelid species were inhibited in their development, while D. caveatum is not inhibited by its own compound(s). D. caveatum has evolved a predation strategy to exploit other members of its genus, including mechanisms of developmental inhibition and specific phagocytosis.     

Identification and analysis of Dictyostelium actin genes,a family of moderately repeated genes

Plasmid M6 has been shown to contain sequences complementary to two related abundant mRNA species which differ in length by 100 nucleotides and code for Dictyostellum actin. M6 complementary RNA was isolated by hybridization to immobilized M6 DNA and translated in vitro. The product is identical to major forms of in vivo labeled actin in both mobility on two-dimensional gels and two-dimensional fingerprints of tryptic peptides. Both plasmid M6 and a second plasmid complementary to the actin mRNA complementary region in M6, pDd actin 2 (McKeown et al., 1978), direct the synthesis in minicells of a number of similar polypeptides that are not seen in minicells containing other recombinant plasmids. Three of these polypeptides are similar in two-dimensional gel mobility to Dictyostelium actin and bind to DNAase I agarose.The repetition frequency of isolated restriction fragments from actin mRNA complementary plasmid M6 has been examined. The data from two different experimental approaches (DNA excess hybridizations using plasmid DNA as probe, and hybridization of plasmid probe to DNA blot filters of restriction enzyme-digested Dictyostelium DNA) indicate that the mRNA complementary region is reiterated 15–20 times. When an actin cDNA probe is used in the same experiments, the results suggest that the entire coding region is reiterated.When the two major actin mRNA species are separated and independently translated, each appears to code for one of the two major actin species. The results suggest that there are at least two different functional genes, and possibly more, for Dictyostelium actin.     

RNA metabolsim during vegetative growth and morphogenesis of the cellular slime mold Dictyostelium discoideum

During vegetative growth of the cellular slime mold Dictyostelium discoideum, RNA is rapidly labeled by radioactive precursor and both the 25 S and the 17 S ribosomal RNA species appear in the cytoplasm 6–7 min after the onset of labeling. Thirty minutes after further incorporation of radioactive RNA precursors has been blocked, less than 10% of the label in RNA is associated with the nuclear fraction. After aggregation of the slime mold amoebae, RNA appears in the cytoplasm at a reduced rate, the small ribosomal subunit appearing in the cytoplasmic fraction more slowly than the larger ribosomal subunit. Some labeled RNA remains in the nuclei of developing cells long after the incorporation of ³H-uridine is blocked.     

Mechano-chemical signaling maintains the rapid movement of Dictyostelium cells

The survival of Dictyostelium cells depends on their ability to efficiently chemotax, either towards food or to form multicellular aggregates. Although the involvement of Ca2+ signaling during chemotaxis is well known, it is not clear how this regulates cell movement. Previously, fish epithelial keratocytes have been shown to display transient increases in intracellular calcium ([Ca2+]i) that are mediated by stretch-activated calcium channels (SACs), which play a role in retraction of the cell body [J. Lee, A. Ishihara, G. Oxford, B. Johnson, and K. Jacobson, Regulation of cell movement is mediated by stretch-activated calcium channels. Nature, 1999. 400(6742): p. 382-6.]. To investigate the involvement of SACs in Dictyostelium movement we performed high resolution calcium imaging in wild-type (NC4A2) Dictyostelium cells to detect changes in [Ca2+]i. We observed small, brief, Ca2+ transients in randomly moving wild-type cells that were dependent on both intracellular and extracellular sources of calcium. Treatment of cells with the SAC blocker gadolinium (Gd3+) inhibited transients and decreased cell speed, consistent with the involvement of SACs in regulating Dictyostelium motility. Additional support for SAC activity was given by the increase in frequency of Ca2+ transients when Dictyostelium cells were moving on a more adhesive substratum or when they were mechanically stretched. We conclude that mechano-chemical signaling via SACs plays a major role in maintaining the rapid movement of Dictyostelium cells.     

Reaction-diffusion waves of actin filament polymerization/depolymerization in Dictyostelium pseudopodium extension and cell locomotion

Cell surface movements and the intracellular spatial patterns and dynamics of actin filament (F-actin) were investigated in living and formalin-fixed cells of Dictyostelium discoideum by confocal microscopy. Excitation waves of F-actin assembly developed and propagated several micrometers at up to 26 microm/min in cells which had been intracellularly loaded with fluorescently labeled actin monomer. Wave propagation and extinction corresponded with the initiation and attenuation of pseudopodium extension and cell advance, respectively. The identification of chemical waves was supported by the ring, sphere, spiral and scroll wave patterns, which were observed in the extensions of fixed cells stained with phalloidin-rhodamine, and by the similar, asymmetrical [F-actin] distribution in wavefronts in living and fixed cells. These F-actin patterns and dynamics in Dictyostelium provide evidence for a new supramolecular state of actin, which propagates as a self-organized, reaction-diffusion wave of reversible F-actin assembly and affects pseudopodium extension. Actin's properties of oscillation and self-organization might also fundamentally determine the nature of the eukaryotic cell's reactions of adaptation, timing and signal response.     

Lysosomal enzymes possess a common antigenic determinant in the cellular slime mold, Dictyostelium discoideum

Antisera have been prepared against two lysosomal enzymes of the cellular slime mold, Dictyostelium discoideum. The two purified enzyme preparations used for immunization, N-acetylglucosaminidase and beta-glucosidase-1, show no cross-contamination with each other and no significant contamination by other lysosomal enzymes. However, antisera raised against either enzyme bind equally well to seven different lysosomal enzymes and show no preference for the enzyme against which they were raised. A total of 10 different antisera have been examined and all show similar results. Preadsorption of antisera with either purified enzyme removes all antibody activity against the other enzyme. Evidence is presented which indicates that the same species of antibodies are responsible for the precipitation of seven lysosomal enzymes. These data are discussed in terms of the proposal that the antigen that is shared by the lysosomal enzymes is a post-translational modification of the enzyme proteins. We have sought to further characterize the distribution of this common antigen among cellular proteins. We show that N-acetylglucosaminidase and beta-glucosidase-1 represent less than 5% of the total common antigen containing proteins in the cell. Precipitation of 35S-labeled cellular proteins from vegetative cells indicates that as much as 15-30% of the total cell protein may possess the common antigen. Preadsorption experiments confirm that all of the proteins immunoprecipitated in these experiments are recognized by the same antibodies that precipitate the lysosomal enzyme activities. Most of the labeled proteins are secreted into the medium along with the lysosomal enzyme activities during axenic growth. During the developmental phase of the life cycle of Dictyostelium, the total amount of the common antigen decreases about 2-fold relative to total cell protein. However, the synthesis of antigenic proteins continues throughout most of development.     

Novel cellular tracks of migrating Dictyostelium cells

After Dictyostelium cells were settled on a coverslip and allowed to migrate freely on the surface, they were stained with fluorescently labeled Concanavalin A. Tracks with distinct patterns that consist of dots and short fibers were observed behind the cells. In this study, we refer to these tracks as "cellular tracks", CTs for short. We characterized the biological effect of CTs on cell behavior and development. CTs decreased the strength of cell-substratum adhesion, increased the velocity of cell migration, but did not affect growth of cells. CTs also promoted cell aggregation. When pre-aggregation cells touched the CTs of other cells, they avoided or orthogonally crossed them, but did not migrate along them. These observations suggest that the CTs of pre-aggregation cells prompts cells to disperse uniformly on substratum and may enable cells to sense cell density. On the other hand, when aggregation-competent cells touched the CTs of other aggregation-competent cells, a half of them migrated along the CTs. Pre-aggregation cells did not migrate along the CTs of aggregation-competent cells. The CTs of aggregation-competent cells may help the cells to aggregate toward the aggregation center.     

Chemotaxis-mediated scission contributes to efficient cytokinesis in Dictyostelium

Interphase amoeba of Entamoeba invadens are attracted to the furrowing region of a neighboring dividing cell to assist with the division. A seemingly similar behavior has been observed in Dictyostelium discoideum, but in this case, it has not been shown whether the movements were truly directed toward the furrowing region or whether they have any relevance. We thus used myosin II-null cells, which spend more time than wild type cells in cytokinesis, and successfully demonstrated that nearly half of the division events involve the attraction of a neighbor cell to the furrowing region. Cells lacking the beta subunit of the trimeric G protein (Gbeta), which are incapable of chemotaxis, did not show such midwifery. Culturing wild type cells flattened under agarose sheets also slowed the cytokinesis process, and this allowed us to demonstrate that phosphatidylinositol trisphosphate was enriched in the anterior region of midwifing cells, consistent with the view that midwifery in D. discoideum is also chemotaxis. On substrates, while only 3.6% of wild type cells were multinucleate, 8.1% of Gbeta-null cells were multinucleate, and this was reduced to 3.4% when they were surrounded by wild type cells. Conversely, multinucleated wild type cells increased to 6.8% when they were surrounded by Gbeta-null cells. Thus, Gbeta-null cells frequently fail to divide because they cannot assist each other's division and midwifery ensures successful cytokinesis in Dictyostelium discoideum.