Calcium,cellular adhesion and aggregation competence in the cellular slime mold Polysphondylium violaceum

Cellular adhesion in Polysphondylium violaceum is mediated by Ca²⁺ ions. The extent of cell adhesion exhibited by developing P. violaceum is greater in the presence of 0.5 mM Ca²⁺ than in the absence of Ca²⁺. Vegetative amebae exhibit some adhesive properties, although the cellular interactions expressed by vegetative amebae are not as extensive as those exhibited by developing amebae. If the amebae are incubated in the presence of chelators (EGTA or EDTA) cellular adhesion is prevented and the amebae remain as single cells. Vegetative cell adhesion is blocked by 1 mM EGTA, whereas blocking adhesion in developing cells requires 5- to 10-fold greater concentrations of EGTA. The acquisition of developmental adhesive properties occurs even if the amebae are incubated in the presence of EGTA, suggesting that Ca²⁺ is required for interaction between adhesion sites but not for their formation. P. violaceum amebae become aggregationcompetent (aggregate immediately when placed on a solid surface) at the same time that the developmental adhesion sites are expressed, even when incubated in the presence of EGTA. Thus it seems unlikely that cellular adhesion is required to develop aggregation competence.     

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.     

Extracellular cyclic AMP during development of the cellular slime mold Polysphondylium violaceum: comparison of accumulation in the wild type and an aggregation-defective mutant.

Cyclic AMP was synthesized by Polysphondylium violaceum after starvation and during the preaggregation stage of development. Most of the newly synthesized cyclic AMP accumulated in the extracellular medium, with very little change in the intracellular cyclic AMP concentration. The addition of 10(-3) to 10(-6) M exogenous cyclic AMP to starved amoebae caused a 20 to 50% decrease in the number of aggregation centers formed compared with untreated controls. An aggregation-defective mutant of P. violaceum (strain aggA586) excreted or accumulated very little cyclic AMP. Strain aggA586 aggregated normally in the presence of a dialyzable, excreted product (D factor) produced by wild-type amoebae. When the mutant was incubated with D factor, cyclic AMP accumulated in the medium, and the amount accumulated depended on the amount of D factor added to the mutant amoebae.     

Developmental regulation of production of an aggregation-stimulating factor from the cellular slime mold Polysphondylium violaceum

An excreted, dialyzable component(s) produced during development of wild-type Polysphondylium violaceum has been previously shown to stimulate aggregation of aggregateless mutants in the complementation group aggA. Production of this aggregation-stimulating factor, called D factor, is greater during development in liquid culture than during development on a surface. after partial purification of crude D factor using high performance liquid chromatography, multiple species are found that retain the ability to stimulate aggregation of the aggA mutants. The three major components (DfA, DfB, and DfC) show decreasing polarity based on purification using reverse-phase chromatography. The proportion of each component secreted varies, depending on the developmental conditions (surface versus liquid) and the time after starvation when the factors are isolated. Preliminary physical and chemical characterization of the three D factor components suggests that they are related.     

Purification, characterization, and partial structure of D factor from Polysphondylium violaceum

The A component of D factor (DfA) was overproduced during development of wild type Polyspondylium violaceum strain China after starvation in liquid medium. Crude DfA excreted by strain China was partially purified by ultrafiltration using Amicon YM10 and YM2 filters with DfA extracted from the filtrate by absorption onto a preparative grade C-18 resin. The concentrated material was further purified on a C-18 analytical column using both acetonitrile:water and methanol:water gradients. This highly purified fraction was a single component with a final specific activity of greater than 10(6) units per mg dry weight. Purified DfA is red having a broad visible absorbance at 500 nm and a ultraviolet (uv) absorbance at 290-300 nm. The red chromophore is sensitive to pH and to oxidation-reduction. 1H and 13C nmr studies with purified DfA indicate that it is a C11 compound with both polar and non-polar regions. The non-polar region has been identified as a hexanone and is the same as the side chain of DIF from Dictyostelium discoideum. Purified DfA has been used in studies with the D factor non-producing mutant, tsg-119 cyc-1 aggA586 (A586), to show that neither production of glorin nor chemotactic sensitivity to glorin are affected by D factor. However, founder cells develop in A586 mutant populations only after addition of D factor. These data suggest that DfA may be necessary for induction of aggregate formation by aggregation-competent amoebae.     

Chemotactic response of wild-type and aggregation-defective mutants of Polysphondylium violaceum

Abstract. The aggregation-specific chemoattractant for Polysphondylium violaceum is N-propionyl-γ-L-glutamyl-L-ornithine-δ-lactam ethyl ester, or glorin. Wild-type amoebae allowed to develop in liquid culture acquire increased ability to respond to glorin shortly after starvation, i.e., just prior to the time they become aggregation competent. Similarly, as development proceeds, the amoebae show decreased sensitivity to folic acid, but they show almost no response to cyclic AMP at any time during their development in liquid culture. The optimum concentrations for the chemotactic response are 10^-8 M for glorin and 10^-5–10^-6 M for folic acid. A class of aggregation-defective mutants, aggA , will not aggregate in the absence of an excreted pheromone, D factor. During development in liquid culture in the presence or absence of D factor, these aggA mutants show a chemotactic response similar to that of wild-type amoebae to folic acid and glorin. However, D factor does enhance the chemotactic response of aggA mutants to glorin. In the absence of D factor, mutant amoebae will form fruiting bodies if exposed to a chemotactic gradient of either folic acid or glorin. Under these conditions, the mutant amoebae circumvent the requirement for D factor in order to develop.     

Immunohistochemical Localization of Cyclic GMP in Aggregating Polysphondylium violaceum

Using indirect immunofluorescence technique, it has been possible to localize cyclic GMP in Polysphondylium violaceum cells. The bound cyclic nucleotide is localized throughout the cell during early stages, however, this staining increases and there is marked localization of cyclic GMP in the nuclear areas of the cells when aggregation is in full swing. Over 90% of the cells exhibited intense nuclear staining by 6 h and this decreased to less than 10% by 10 h.     

Acidic protein kinases of Polysphondylium violaceum: Characterization and developmental regulation

Summary The cellular slime mould Polysphondylium violaceum contains two vegetative stage specific acidic (casein) kinases. These two enzymes have been partially purified and their properties investigated. Both utilise casein as their preferred substrate but they can be distinguished in a number of ways, including their responses to spermine, heparin and salt. In addition, they have different affinities for their substrates and different pH activity profiles. It is suggested that they may play a role in a vegetative specific function such as cell division.     

Syngenic Divisions of the Cellular Slime Mold Polysphondylium violaceum*

SYNOPSIS. Mating type analysis of new isolates of Polysphondylium violaceum supports the subdivision of this morphological species into two reproductively isolated breeding groups or syngens. Representatives of both syngens have been identified in soil samples taken from widely separated geographical locations. Intersyngenic cross reactions have been observed in some stocks.     

Analysis of developmentally defective chemical signaling mutants of Polysphondylium violaceum.

Six aggregation-defective mutants of Polysphondylium violaceum dependent on external addition of the pheromone D factor for aggregation were isolated after nitrosoguanidine mutagenesis. With a screening technique based on synergistic development, D-factor-dependent mutants can be separated from other kinds of aggregateless mutants. Genetic complementation analyses of the newly isolated mutants showed them to be mutant at the aggA locus. Individual mutants exhibited different sensitivities to D factor(s), responding maximally over a 300-fold range of concentrations.     

Cytosolic cAMP-dependent protein kinase of Polysphondylium violaceum: developmental regulation and properties

Summary The cellular slime mould Polysphondylium violaceum contains a cAMP-dependent protein kinase resembling the mammalian type I enzyme. The appearance of this enzyme is developmentally regulated. The level of kinase activity is very low in vegetative cell and increases more than tenfold during differentiation.The catalytic subunit of this cAMP-dependent protein kinase has a native molecular weight of 60–80 kDa, an isoelectric point of 5.7 and an apparent K_m for ATP and Kemptide of 50 and 13.4 µM respectively. It is characterised by its sensitivity to a synthetic inhibitor specific for cAMP-dependent protein kinase. The regulatory subunit has a molecular weight of 50 kDa.Abbreviations HEPES N-2-Hydroxyethylpiperazine-N-2-ethane sulphonic acid - EDTA ethylenediamine tetraacetic acid - EGTA ethyleneglycol-bis-(ßaminoethyl ether)-N,N,N,N-tetraacetic acid - SDS sodium dodecyl sulphate     

Alterations of cell-surface carbohydrates during differentiation and development

Expression of many cell-surface carbohydrates is controlled temporally and spatially by developmental programs. This subject is reviewed from 5 viewpoints: structural changes revealed by chemical analysis, cell-surface markers useful for cell identification and separation, core proteins carrying the developmentally regulated carbohydrate chain, glycosyltransferases responsible for the change and the biological meaning of the phenomenon. The differentiation systems covered are mainly early mammalian embryogenesis and the differentiation of blood and nerve cells.     

Serological investigations of cell adhesion in the slime molds,Dictyostelium discoideum,Dictyostelium purpureum,and Polysphondylium violaceum

1. Antibodies to slime molds were produced by injecting D. discoideum and D. purpureum amebas from 48 hour cultures into rabbits. 2. Anti-D. discoideum and anti-D. purpureum sera caused agglutination of homologous amebas from 24 to 26 hour cultures, agglutination of certain heterologous amebas from 30 to 36 hour cultures, and agglutination of all heterologous amebas from 43 to 48 hour cultures. 3. The data show that new surface antigens are formed in cultures after 26 hours and it is suggested that the new antigens are concerned with cell adhesion. 4. The probable role of surface antigens in the interaction of cells of different species of slime molds was discussed.     

DWnt4 and wingless elicit similar cellular responses during imaginal development

Wnt genes encode evolutionarily conserved secreted proteins that provide critical functions during development. Although Wnt proteins share highly conserved features, they also show sequence divergence, which almost certainly contributes to the variety of their signaling activities. We previously reported that DWnt4 and wingless (wg), two divergent clustered Wnt genes, can have either antagonist or distinct functions during Drosophila embryogenesis. Here we provide evidence that both genes can elicit similar cellular responses during imaginal development. Ectopic expression of DWnt4 along the anterior/posterior (A/P) boundary of imaginal discs alters morphogenesis of adult appendages. In the wing disc, DWnt4 phenocopies ectopic Wg activity by inducing notum to wing transformation, suggesting similar signaling capabilities of both molecules. In support of this, we demonstrate that DWnt4 can rescue wg loss-of-function phenotypes in the antenna and haltere and is able to substitute for Wg in wing field specification. We also show that both genes are transcribed in overlapping domains in imaginal discs, suggesting that DWnt4 may cooperate with wg during limb patterning.     

Synthesis of macromolecules during microcyst germination in the cellular slime mold Polysphondylium pallidum

Microcyst germination in the cellular slime mold Polysphondylium pallidum is a useful model for studying macromolecular changes necessary for or coincident with the transition from one cell type (cyst) to another (amoebae). Protein synthesis starts soon after cysts are incubated under permissive conditions, as evidenced by the incorporation of precursors and the appearance of polysomes. Sodium dodecyl sulfate-polyacrylamide gel analysis of proteins made at intervals during germination shows that protein synthesis is developmentally regulated during this process. RNA synthesis also begins early during germination. Cysts contain polyadenylated RNA that can stimulate the incorporation of radioactive amino acids into protein in an in vitro wheat germ protein synthesizing system. The concentration of poly(A)-containing RNA increases during germination and during inhibition of protein synthesis by cycloheximide.     

Differential expression of mepA, mepC and smtE during growth and development of Microbotryum violaceum

Many fungi require a dimorphic switch from budding to filamentous growth to cause infection. Although the control of dimorphism has been elucidated for organisms such as Saccharomyces cerevisiae and Ustilago maydis, almost nothing is known about the control of mating and dimorphism in Microbotryum violaceum. M. violaceum mepA, mepC and smtE are homologs of genes whose encoded products act as, or interact with, components of the MAPK and cAMP-PKA pathways, conserved pathways that regulate mating and dimorphism in other fungi. A comparison of gene expression under various in vitro conditions was superimposed on a comparison of in vitro vs. in planta expression to yield a more complete picture of the expression of these genes in M. violaceum during fungal development. For the most part the expression of these genes was highest on low ammonium, intermediate for mated and in planta, and lowest on rich medium. As expected, under conditions of low ammonium, expression of the M. violaceum ammonium permease genes mepA and mepC mirrors that of S. cerevisiae MEP2 and U. maydis ump2. An intriguing possibility is that MepA is a sensor to signal when conditions are conducive for mating. The upregulation of smtE, which encodes a PAK kinase, suggests that the MAPK pathway regulates, at least partially, mating and might be linked to ammonium sensing/transport in M. violaceum.     

An increase of cAMP-dependent protein kinase during development in Polysphondylium pallidum

Polysphondylium pallidum is a cellular slime mold in which, unlike in Dictyostelium discoideum, cAMP is not the chemotactic agent. The occurrence of a cAMP-dependent protein kinase in D. discoideum was demonstrated earlier and we suggested that it may mediate the intracellular effects of cAMP on the development of the organism, particularly since an increase in the amount of the enzyme during development was noted. In D. discoideum cAMP plays a dual role insofar as it serves both as chemotactic agent and as second messenger; it was of interest therefore, to determine whether a cAMP-dependent protein kinase occurred in P. pallidum. We found a cAMP-dependent protein kinase in P. pallidum using Kemptide as substrate. The regulatory subunit of the enzyme has an apparent molecular weight of 41,000 and seems to be similar in its properties with that isolated earlier from D. discoideum. The cAMP-dependent protein kinase catalytic subunits from the two species are also similar. Furthermore, there is a developmentally regulated, parallel, two- to threefold increase in the two subunits of the cAMP-dependent protein kinase in P. pallidum. The increase occurs before aggregates are formed. These findings are compatible with a role of the intracellular cAMP and of the cAMP-dependent protein kinase in the development of P. pallidum.     

Heat shock response in a cellular slime mold,Polysphondylium pallidum

Cells of Polysphondylium pallidum were exposed to a heat shock by raising the temperature from 25 to 31°C. A set of four major polypeptides of approximate molecular weights 105,000, 87,000, 74,000, and 33,000 incorporated [1-¹⁴C]acetate when pulse labeled during the first hour after heat shock. The response resembles the heat shock response of Drosophila in occurring in cells at different stages of development (early in aggregation, late in aggregation, and during microcyst formation) and in being triggered by a threshold high temperature rather than a minimal change in temperature.