Unusual polyamines in slime molds Physarum polycephalum and Dictyostelium discoideum

We analyzed the cellular contents of not only major polyamines but also minor polyamines in slime molds Physarum polycephalum and Dictyostelium discoideum. The presence of putrescine and spermidine in either plasmodia or myxamoebae of these molds as major polyamines was confirmed. In addition to these polyamines, appreciable amounts of 1,3-diaminopropane were detected in P. polycephalum and D. discoideum. Cadaverine and sym-homospermidine were detected in P. polycephalum even when the slime mold was cultured in a chemically defined growth medium. Spermine was not detected when these molds were grown in synthetic media. Other "unusual" polyamines such as norspermidine, norspermine, thermospermine, aminopropylcadaverine, and canavalmine were not detected in either mold.     

The role of membrane lectins in Dictyostelium discoideum aggregation as ascertained by specific univalent antibodies against discoidin I

Antibodies against pure discoidin I have been used as a tool to ascertain the role of this lectin in aggregation of Dictyostelium discoideum. Discoidin I is widely expressed over the cell surface of aggregation-competent AX-2 cells, as ascertained by indirect immunofluorescence with specific (antidiscoidin I) antibodies. Univalent antidiscoidin I antibodies (Fab fragments) inhibit the aggregation-specific intercellular adhesion of D discoideum AX-2 cells in an in vitro assay. This inhibition depends on antibody concentration and cell density; a 50% inhibition of cell aggregation was obtained at antidiscoidin I Fab concentration of 4.5 mg/ml and 1 X 10(6) cells/ml. Aggregation and morphogenesis on solid support is also effectively inhibited when AX-2 cells are starved in the presence of antidiscoidin I Fab fragments. The inhibition of morphogenesis is also dose dependent and more effective than in the in vitro assay. No inhibition of aggregation either in the in vitro assay or on morphogenesis on solid support was observed with preimmune Fab fragments at any of the concentrations tested (up to 9.6 mg/ml).     

The timing of cell-type-specific differentiation in Dictyostelium discoideum

We have used two-dimensional gel electrophoresis to identify over 30 proteins which are specific to one or other of the two cell types of Dictyostelium discoideum, either at the slug stage or in mature fruiting bodies. Our results support the idea that there is a continuous developmental program that begins in prespore cells at the hemispherical mound stage (10-12 hr) and results in spore differentiation (24 hr). Prestalk differentiation, on the other hand, appeared largely unrelated to stalk differentiation, which was first detectable at the onset of culmination (18 hr). We have also used this approach to study the differentiation of stalk-only mutants and have found that the cells can switch from spore to stalk differentiation as late as 2 hr before the end of the wild-type developmental program.     

Inhibition of cell differentiation and cell cohesion by tunicamycin in Dictyostelium discoideum

The effects of tunicamycin on protein glycosylation and cell differentiation were examined during early development of Dictyostelium discoideum. Tunicamycin inhibited cell growth reversibly in liquid medium. At a concentration of 3 μg/ml, tunicamycin completely inhibited morphogenesis and cell differentiation in developing cells. These cells remained as a smooth lawn and failed to undergo chemotactic migration. The expression of EDTA-resistant contact sites was also inhibited. The inhibition by tunicamycin was reversible if cells were washed free of the drug within the first 10 hr of incubation. After 12 hr of development, cells were protected from the drug by the sheath. When cells were treated with tunicamycin during the first 10 hr of development, incorporation of [³H]mannose and [³H] fucose was inhibited by approximately 75% within 45 min while no significant inhibition of [³H]leucine incorporation was observed during the initial 3 hr of drug treatment. The inhibition of protein glycosylation was further evidenced by the reduction in number of glycoproteins “stained” with ¹²⁵I-labelled con A. A number of developmentally regulated high-molecular-weight glycoproteins, including the contact site A glycoprotein (gp80), were undetectable when cells were labelled with [³H]fucose in the presence of tunicamycin. It is therefore evident that glycoproteins with N-glycosidically linked carbohydrate moieties may play a crucial role in intercellular cohesiveness and early development of D. discoideum.     

盘基网柄菌细胞分化及细胞比例的调控

本文综述了盘基网柄菌(Dictyosteliumdiscoideum)发育过程中调控细胞分化及细胞比例的一些信号分子,包括分化诱导因子(DIF-1、SDF-2)、糖原合成酶激酶(GSK-3)、环状亮氨酸拉链蛋白(rZIP)等,介绍了这些信号分子的功能及其作用机制。     

Initiation of multicellular differentiation in Dictyostelium discoideum is regulated by coronin A

Many biological systems respond to environmental changes by activating intracellular signaling cascades, resulting in an appropriate response. One such system is represented by the social amoeba Dictyostelium discoideum. When food sources become scarce, these unicellular cells can initiate a cAMP-driven multicellular aggregation program to ensure long-term survival. On starvation, the cells secrete conditioned medium factors that initiate cAMP signal transduction by inducing expression of genes such as cAMP receptors and adenylate cyclase. The mechanisms involved in the activation of the first pulses of cAMP release have been unclear. We here show a crucial role for the evolutionarily conserved protein coronin A in the initiation of the cAMP response. On starvation, coronin A–deficient cells failed to up-regulate the expression of cAMP-regulated genes, thereby failing to initiate development, despite a normal prestarvation response. Of importance, external addition of cAMP to coronin A–deficient cells resulted in normal chemotaxis and aggregate formation, thereby restoring the developmental program and suggesting a functional cAMP relay in the absence of coronin A. These results suggest that coronin A is dispensable for cAMP sensing, chemotaxis, and development per se but is part of a signal transduction cascade essential for system initiation leading to multicellular development in Dictyostelium.     

On induction of cell differentiation by cyclic AMP pulses in Dictyostelium discoideum

Repeated pulses of cyclic AMP, applied at intervals of 5 min, efficiently induced differentiation in cells of agip 53, a morphogenetic mutant of Dictyostelium discoideum, strain Ax-2. In contrast, pulses applied at intervals of 2 min did not induce cell differentiation. To analyze this phenomenon the hydrolysis of cyclic AMP between the pulses as well as the effect of the pulses on the intracellular concentrations of cyclic GMP were investigated. Experiments performed in the presence of added cyclic AMP was not the reason of the inefficiency of the pulses applied with a 2-min rhythm. Cyclic AMP pulses applied at intervals of 2 min induced discrete increases of the cyclic GMP concentration. Limited time resolution at the level of cyclic GMP cannot account for the inefficiency of the 2-min pulses.     

On induction of cell differentiation by cyclic AMP pulses in Dictyostelium discoideum

Repeated pulses of cyclic AMP, applied at intervals of 5 min, efficiently induced differentiation in cells of agip 53, a morphogenetic mutant of Dictyostelium discoideum, strain Ax-2. In contrast, pulses applied at intervals of 2 min did not induce cell differentiation. To analyze this phenomenon the hydrolysis of cyclic AMP between the pulses as well as the effect of the pulses on the intracellular concentration of cyclic GMP were investigated. Experiments performed in the presence of added cyclic AMP phosphodiesterase revealed that incomplete hydrolysis of cyclic AMP was not the reason for the inefficiency of the pulses applied with a 2-min rhythm. Cyclic AMP pulses applied at intervals of 2 min induced discrete increases of the cyclic GMP concentration. Limited time resolution at the level of cyclic GMP cannot account for the inefficiency of the 2-min pulses.Based on material presented at the Symposium Intercellular Communication Stuttgart, September 16–17, 1982     

Extracellular cyclic AMP-phosphodiesterase accelerates differentiation in Dictyostelium discoideum.

Extracellular cyclic AMP-phosphodiesterase accelerates the development of aggregation competence in Dictyostelium discoideum when present during the preaggregation stage. The effect on development appears to depend only on hydrolysis of extracellular cyclic AMP and not on other properties of the phosphodiesterase molecule. Extracellular cyclic AMP-phosphodiesterase, as a promoter of differentiation, acts mainly throughout the first half of interphase. Our evidence supports the proposal that cyclic AMP oscillations control the rate and possibly the initiation of development. Since extracellular cyclic AMP-phosphodiesterase acts from the beginning of interphase cyclic AMP oscillations may also occur from early interphase, at least in the presence of this enzyme. This would imply that the cyclic AMP oscillator is a determinant, but not a product, of the developmental programme.     

Monoclonal antibodies specific for stalk differentiation in Dictyostelium discoideum

We have produced two monoclonal antibodies specific to the stalk cells of Dictyostelium discoideum fruiting bodies. Both monoclonal antibodies react with high molecular weight proteins previously found to be stalk-specific by two-dimensional gel analysis. One antibody (JAb 1) is specific for a single protein of apparent molecular weight 310 000 which first appears when overt stalk differentiation begins at 20 h. The other monoclonal antibody (JAb 2) is also stalk-specific, though earlier in development it binds to proteins extracted from both prestalk and prespore cells of the migrating slug. It reacts with two proteins in stalks, one of apparent molecular weight 430 000 which is first detected during tip formation at 12 h and a lower molecular weight protein (310 000) detected from 20 h. Although several markers are available for the investigation of prespore/spore differentiation there is a distinct lack of suitable prestalk/stalk markers. The monoclonal antibodies described here are highly specific stalk markers and should prove useful in the study of cell proportioning and terminal differentiation.     

Identification of new differentiation inducing factors from Dictyostelium discoideum

Developing Dictyostelium discoideum amoebae form a stalked fruiting body in which individual cells differentiate into either stalk cells or spores. The major known inducer of stalk cell differentiation is the chlorinated polyketide DIF-1 (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one); however a mutant blocked in the terminal step of DIF-1 biosynthesis still produces one of the prestalk cell subtypes - the pstA cells - as well as some mature stalk cells. We therefore searched for additional stalk cell-inducing factors in the medium supporting development of this mutant. These factors were purified by solvent extraction and HPLC and identified by mass spectroscopy and NMR. The mutant lacked detectable DIF-2 and DIF-3 (the pentanone and deschloro homologues of DIF-1) but four major stalk cell-inducing activities were detected, of which three were identified. Two compounds were predicted intermediates in DIF-1 biosynthesis: the desmethyl, and desmethyl-monochloro analogues of DIF-1 (dM-DIF-1 and Cl-THPH, respectively), supporting the previously proposed pathway of DIF-1 biosynthesis. The third compound was a novel factor and was identified as 4-methyl-5-pentylbenzene-1,3-diol (MPBD) with the structure confirmed by chemical synthesis. To investigate the potential roles of these compounds as signal molecules, their effects on morphological stalk and spore differentiation were examined in cell culture. All three induced morphological stalk cell differentiation. We found that synthetic MPBD also stimulated spore cell differentiation. Now that these factors are known to be produced and released during development, their biological roles can be pursued further.     

Cell differentiation in Dictyostelium discoideum controls assembly of protein-linked glycans

The prestalk and prespore cells from the Dictyostelium discoideummulticellular slug stage of development differ in assembly ofglycoconjugates. Prespore cells are 2- to 3-fold more activethan prestalk cells in the assembly of N-linked glycans and20-fold more active in their fucosylation. Only prespore cellssynthesize an O-linked glycan consisting in part of Fuc -linkedto N-acetylglucosamine. Incorporation of fucose, glucosamine,mannose and galactose into large pronase-resistant glycoconjugateswas almost exclusively into prespore cells. Such glucosamine-labelledglycoconjugates resist fragmentation by ß-eliminationand include a glycoantigen dependent on the modB genetic locus.In contrast, large fucose-labelled glycoconjugates consistedof multiple, small, O-linked oligosaccharides on carrier peptides.The spore coat protein SP96 has several fucosylated O-linkedoligosaccharides, one of which correlates with a fucose epitopepreviously shown to localize in prespore vesicles and the outerlayer of the spore coat. Dictyostelium discoideum glycoconjugates glycoproteins prespore prestalk     

Role of Cell Sorting in Pattern Formation in Dictyostelium discoideum

To examine the relationship between cell sorting and cell differentiation in the development of Dictyostelium discoideum , labeled cells grown in the absence of glucose [G(–) cells] and unlabeled cells grown in its presence [G(+) cells] were mixed and either allowed to undergo normal morphogenesis or cultured under submerged conditions. Changes in the distributions within a cell aggregate of labeled cells and cells stained with the conjugated antispore serum (prespore cells) were followed on the same sections by the methods of autoradiography and immunohistochemistry. In normal morphogenesis, differentiation of prespore cells apparently initiated and proceeded coincident with sorting out between G(+) and G(–) cells, during formation of a standing slug. By contrast, within an aggregate formed under submerged conditions, prespore cells began to differentiate long before G(+) and G(–) cells were sorted out, indicating that the cell sorting is not a prerequisite for the cell differentiation. The sorting out, however, brought about an accumulation of prespore cells in a hemisphere, thus producing a prestalk-prespore pattern within the aggregate.     

Dictyostelium discoideum: a model system for differentiation and patterning

In Dictyostelium, development begins with the aggregation of free living amoebae, which soon become organized into a relatively simple organism with a few different cell types. Coordinated cell type differentiation and morphogenesis lead to a final fruiting body that allows the dispersal of spores. The study of these processes is having increasing impact on our understanding of general developmental mechanisms. The availability of biochemical and molecular genetics techniques has allowed the discovery of complex signaling networks which are essential for Dictyostelium development and are also conserved in other organisms. The levels of cAMP (both intracellular and extracellular) play essential roles in every stage of Dictyostelium development, regulating many different signal transduction pathways. Two-component systems, involving histidine kinases and response regulators, have been found to regulate intracellular cAMP levels and PKA during terminal differentiation. The sequence of the Dictyostelium genome is expected to be completed in less than two years. Nevertheless, the available sequences that are already being released, together with the results of expressed sequence tags (ESTs), are providing invaluable tools to identify new and interesting genes for further functional analysis. Global expression studies, using DNA microarrays in synchronous development to study temporal changes in gene expression, are presently being developed. In the near future, the application of this type of technology to the complete set of Dictyostelium genes (approximately 10,000) will facilitate the discovery of the effects of mutation of components of the signaling networks that regulate Dictyostelium development on changes in gene expression.