Dual Effects of cAMP on the Stability of Prespore Vesicles and 8-bromo cAMP-enhanced Maturation of Spore and Stalk Cells of Dictyostelium discoideum

It is well known that interconversion between prestalk and prespore cells occurs in 3-dimensional (3–D) isolates of Dictyostelium. The present work was undertaken to examine whether or not the interconversion occurs even in monolayer sheets. The results suggested that in monolayer sheets of either prespore or prestalk cells, the interconversion does not occur. Furthermore, effects of cAMP were examined in relation to the formation or loss of prespore vesicles (PSVs). In monolayer sheets, prespore cells retain their PSVs in the presence of cAMP, though they lose them in its absence. In 3–D masses, however, cAMP induces the conversion into stalk cells, stimulating PSV loss. In the case of prestalk cells, cAMP induces the maturation of prestalk cells to stalk cells in 3–D masses, but it does not induce stalk differentiation in monolayer sheets.
8-Bromo cAMP stimulates the maturation of prespore and prestalk cells into spore and stalk cells, respectively. However, the vegetative and the aggregative cells remain amoeboid even in its presence. These observations suggest that 8-bromo cAMP stimulates the maturation rather than inducing prespore and prestalk differentiation.     

Involvements of a novel protein, DIA2, in cAMP signaling and spore differentiation during Dictyostelium development

Abstract The novel gene dia2 (differentiation-associated gene 2) was originally isolated as a gene expressed specifically in response to initial differentiation of Dictyostelium discoideum Ax-2 cells. Using dia2^AS cells in which the dia2 expression was inactivated by the antisense RNA method, DIA2 protein was found to be required for cAMP signaling during cell aggregation. During late development, the DIA2 protein changed its location from the endoplasmic reticulum (ER) to prespore-specific vacuoles (PSVs) that are specifically present in prespore cells of the slug. In differentiating prestalk cells, however, DIA2 was found to be nearly lost from the cells. Importantly, exocytosis of PSVs from prespore cells and the subsequent spore differentiation were almost completely impaired in dia2^AS cells. In addition, spore induction by externally applied 8-bromo cAMP was significantly suppressed in dia2^AS cells. Taken together, these results strongly suggested that DIA2 might be closely involved in cAMP signaling and spore differentiation as well as in the initiation of differentiation during Dictyostelium development.     

Involvement of the TRAP-1 homologue, Dd-TRAP1, in spore differentiation during Dictyostelium development

Tumor necrosis factor receptor-associated protein 1 (TRAP1) is a member of the molecular chaperone HSP90 (90-kDa heat shock protein) family. We have previously demonstrated that Dictyostelium discoideum TRAP1 (Dd-TRAP1) synthesized at the vegetative growth phase is retained during the whole course of D. discoideum development, and that at the multicellular slug stage, it is located in prespore-specific vacuoles (PSVs) of prespore cells as well as in the cell membrane and mitochondria. Thereupon, we examined the function of Dd-TRAP1 in prepore and spore differentiation, using Dd-TRAP1-knockdown cells (TRAP1-RNAi cells) produced by the RNA interference method. As was expected, Dd-TRAP1 contained in the PSV was found to be exocytosed during sporulation to constitute the outer-most layer of the spore cell wall. In the TRAP1-RNAi cells, PSV formation and therefore prespore differentiation were significantly impaired, particularly under a heat stress condition. Although the TRAP1-RNAi cells formed apparently normal-shaped spores with a cellulosic wall, the spores were less resistant to heat and detergent treatments, as compared with those of parental MB35 cells derived from Ax-2 cells. These findings strongly suggest that Dd-TRAP1 may be closely involved in late development including spore differentiation, as well as in early development as realized by its induction of prestarvation response.     

Proteomics opens doors to the mechanisms of developmentally regulated secretion

The program of multicellular development in Dictyostelium discoideum culminates with the assembly of a rugged, environmentally resistant spore coat around each spore cell. After synthesis, the proteins that will constitute the coat are stored in prespore vesicles (PSVs) until an unknown developmental signal triggers the PSVs to move to the cell surface where they fuse with the plasma membrane and secrete their cargo by exocytosis. These events occur synchronously in 80% of the cells in each developing multicellular aggregate, and thus the system offers a unique opportunity to study the developmental regulation of protein secretion in situ. Proteomic analysis of purified PSVs identified many of the constituent proteins, which in turn has lead to novel hypotheses and new experimental avenues regarding the molecular mechanisms regulating secretion from the PSVs.     

The prespore vesicles of Dictyostelium discoideum. Purification, characterization, and developmental regulation

The coordinate fusion of the prespore vesicles (PSVs) with the plasma membrane at the terminal stage of spore differentiation in Dictyostelium discoideum is an important example of developmentally regulated protein secretion. However, little is known about the composition of the vesicles, the molecular signals regulating secretion, or the mechanics of the membrane fusion. Taking a biochemical approach, we purified PSVs from different developmental stages. These preparations are highly enriched for their specific cargo of spore coat proteins while devoid of markers for other cellular compartments. Electron microscopic observations show that the PSV preparations are homogenous, with the soluble spore coat protein PsB/SP85 distributed throughout the lumen and the acid mucopolysaccharide localized in the central core. During development the PSVs increase in size and density concomitant with an increase in their protein cargo. The PSVs contain approximately 80 proteins, and we have identified a PSV-specific GTP-binding protein that may be involved in regulating vesicle fusion. The PSVs are not clathrin-coated and do not contain the SpiA spore coat protein. The PSV preparations are ideal for a global proteome analysis to identify proteins involved in signal reception, vesicle movement, docking, and fusion in this developmentally regulated organelle.     

Proteomic analysis of a developmentally regulated secretory vesicle

Secretion of spore coat proteins from the prespore secretory vesicles (PSVs) in Dictyostelium discoideum is a signal mediated event that underlies terminal cell differentiation, and represents an important case of developmentally regulated secretion. In order to study the biochemical mechanisms that govern the regulated fusion of the PSVs with the plasma membrane and the subsequent secretion of their cargo, we purified this organelle from prespore cells. Analysis of protein extracts of highly purified PSVs indicated that, in addition to the cargo of structural spore coat proteins, many more proteins are associated with the PSVs. Their identification is paramount to the understanding of the mechanism of regulated secretion in this system. In this study we have taken the first comprehensive proteomic approach to the analysis of an entire, previously uncharacterized, organelle, with the goal of identifying the major proteins associated with the PSVs. We show that in addition to the structural spore coat proteins, the PSVs contain the enzymes needed for proper spore coat assembly (thioredoxin 2 and 3), regulatory proteins which we predict receive and transduce the developmental signal for secretion (rab7 GTPase, PI-3 kinase, NDP kinase and the calcium binding proteins calfumirin-1 and calreticulin) as well as proteins that interact with the cytoskeleton to mediate movement of the PSVs to the plasma membrane (actin binding proteins coactosin and profilin 1). In addition, the results suggest that proteins can play multiple roles in the cell, and that protein function can be dictated in part by subcellular localization. The identification of the PSV proteins is allowing us to develop testable hypotheses about the roles of these proteins within the functional context of developmentally regulated secretion.     

RasG protein accumulation occurs just prior to amoebae emergence during spore germination in Dictyostelium discoideum

Abstract RasG protein levels in dormant and germinating spores of Dictyostelium discoideum strains JC1 and SG1 were estimated by Western blotting. Ras Glevels were very low in dormant spores and remained low during the lag period, regardless of whether spores were heat activated or treated with autoactivator during the early stages of spore germination. RasG levels increased late during spore swelling just prior to the emergence stage of germination. These data are consistent with a requirement for RasG during vegetative growth.     

CONCANAVALIN A-INDUCED AGGLUTINATION AND BINDING OF CON A TO THE DIFFERENTIATING CELLS OF DICTYOSTELIUM DISCOIDEUM

Changes in agglutinability of Dictyostelium discoideum cells with Concanavalin A (Con A) during the course of development were investigated. The agglutinability of the cells was assayed under conditions where no spontaneous cell agglutination occurred. It was found that there was a progressive decrease in Con A-induced agglutinability during development: a decrease to half from exponentially growing cells to preaggregation cells, and to sixth in disaggregated slug cells. Pronase-BAL treatment of preaggregation cells did not enhance their agglutinability with Con A. The amounts of sites available for binding Con A were determined with preaggregation and slug cells. Cells were incubated at 4°C and in the presence of NaN₃ to avoid possible endocytosis of Con A. No significant differences in numbers of Con A-binding sites per unit area of cell surface was detected among preaggregation cells, those treated with pronase and BAL and cells disaggregated from slugs by similar treatment. It was thus concluded that the decrease in Con A-induced agglutinability during development is not attributable to changes in the numbers of Con A-binding sites.     

Structural and biochemical aspects of cell motility in amebas of Dictyostelium discoideum

Amebas of Dictyostelium discoideum contain both microfilaments and microtubules. Microfilaments, found primarily in a cortical filament network, aggregate into bundles when glycerinated cells contract in response to Mg-ATP. These cortical filaments bind heavy meromyosin. Microtubules are sparse in amebas before aggregation. Colchicine, griseofulvin, or cold treatments do not affect cell motility or cell shape. Saltatory movement of cytoplasmic particles is inhibited by these treatments and the particles subsequently accumulate in the posterior of the cell. Cell motility rate changes as Dicytostelium amebas go through different stages of the life cycle. Quantitation of cellular actin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the quantity of cellular actin changes during the life cycle. These changes in actin are directly correlated with changes in motility rate. Addition of cyclic AMP to Dictyostelium cultures at the end of the feeding stage prevents a decline in motility rate during the preaggregation stage. Cyclic AMP also modifies the change in actin content of the cells during preaggregation.     

Localization of fluorescent substances in the cellular slime moldDictyostelium discoideum cells during growth and development

The localization of fluorescent substance was observed microscopically in livingDictyostelium discoideum cells. The fluorescence was localized in the vacuoles of the vegetative cells. The fluorescent vacuoles were not observed in the dead cells. The fluorescent vacuoles in the cytoplasm were lost in starved cells which are able to form an aggregate and to differentiate. The fluorescent vacuoles were not lost but decreased slightly in the cytoplasm of full grown cells and of cells grown in liquid nutrient medium for an extended period of time (stationary phase cells). On a solid substratum, fluorescent vacuoles were also lost from the cells, where the vegetative cells aggregate and form a slug-shaped mass of cells. The whole slug showed homogeneous fluorescence. In a finally constructed fruiting body, the spore mass showed fluorescence. In a spore mass, the fluorescence was not observed in the spores but in the interspore space of the spore mass. It is suggested that vegetative cells secrete fluorescent substance into the inter-cellular space in the mass of cells during development.     

The Effect of Proteases on Gene Expression and Cell Differentiation in Dictyostelium discoideum

We have examined the effects of chymotrypsin or pronase on the differentiation of monolayers of Dictyostelium discoideum amoebae developing in the presence of 1–5 mM cyclic AMP. Using sporogenous mutants, which are capable of forming both spores and stalk cells under these conditions, we have observed that low concentrations of either protease selectively inhibit a late step of spore formation. Higher levels of the proteases act at an earlier time and by a distinct mechanism to reduce the accumulation of the prespore cell specific enzyme UDP galactose polysaccharide transferase while not affecting the appearance of glycogen phosphorylase. The latter is present in both prestalk and prespore cells.     

Diphospho-myo-inositol phosphates during the life cycle of Dictyostelium and Polysphondylium.

The intracellular amounts of diphospho-myo-inositol phosphates and InsP6 were determined in Dictyostelium discoideum AX2 throughout the life cycle, including exponential growth, starvation, differentiation, sporulation and spore germination. Similar experiments were performed with the closely related species Polysphondylium pallidum under conditions resulting in microcyst formation. A distinct accumulation of these compounds is observed during the early starvation phase of the cell population before the onset of the actual differentiation program. When exponentially growing D. discoideum cells were shifted to starvation conditions, a 25-fold accumulation of 5,6-bis-PP-InsP4 within 3 h was observed. In P. pallidum, the 5,6-bis-PP-InsP4 pool rises around 20-fold within 8 h during the formation of microcysts from vegetative cells. Finally, the diphosphoinositol phosphates are deposited in spores or microcysts and are degraded when spores or microcysts germinate at low cell density.     

Bimodal distribution of motility and cell fate in Dictyostelium discoideum

Pre-starvation amoebae of Dictyostelium discoideum exhibit random movements. Starved cells aggregate by directed movements (chemotaxis) towards cyclic AMP and differentiate into live spores or dead stalk cells. Many differences between presumptive spore and stalk cells precede differentiation. We have examined whether cell motility-related factors are also among them. Cell speeds and localisation of motility-related signalling molecules were monitored by live cell imaging and immunostaining (a) in nutrient medium during growth, (b) immediately following transfer to starvation medium and (c) in nutrient medium that was re-introduced after a brief period of starvation. Cells moved randomly under all three conditions but mean speeds increased following transfer from nutrient medium to starvation medium; the transition occurred within 15 min. The distribution of speeds in starvation medium was bimodal: about 20% of the cells moved significantly faster than the remaining 80%. The motility-related molecules F-actin, PTEN and PI3 kinase were distributed differently in slow and fast cells. Among starved cells, the calcium content of slower cells was lower than that of the faster cells. All differences reverted within 15 min after restoration of the nutrient medium. The slow/fast distinction was missing in Polysphondylium pallidum, a cellular slime mould that lacks the presumptive stalk and spore cell classes, and in the trishanku (triA(-)) mutant of D. discoideum, in which the classes exist but are unstable. The transition from growth to starvation triggers a spontaneous and reversible switch in the distribution of D. discoideum cell speeds. Cells whose calcium content is relatively low (known to be presumptive spore cells) move slower than those whose calcium levels are higher (known to be presumptive stalk cells). Slow and fast cells show different distributions of motility-related proteins. The switch is indicative of a bistable mechanism underlying cell motility.     

THE EFFECTS OF CYCLIC AMP ON DIFFERENTIATION OF A MUTANT DICTYOSTELIUM DISCOIDEUM CAPABLE OF DEVELOPING WITHOUT MORPHOGENESIS

The dev 1510 mutant of Dictyostelium discoideum differs from the wild type in that unaggregated cells are capable of differentiating into either spores or stalk cells depending on the culture conditions (12). Taking advantage of this fact, the effects of cyclic AMP (cAMP) on differentiation of the mutant cells were examined under conditions that prevent normal morphogenesis. In the presence of low concentrations of exogenous cAMP, the cells differentiated into only stalk cells, whereas in the presence of high concentrations they differentiated into only spores. Untreated cells formed stalk cells, but this was inhibited by addition of phosphodiesterase, indicating that it was induced by a low concentration of cAMP which they produced themselves. Cyclic GMP and dibutyryl cAMP also induced spore formation though less effectively, while 5'AMP, ADP and ATP had no effect. During development, the cells increased in sensitivity to cAMP in that spore formation was induced at lower concentration of cAMP after 4 hr of starvation. Treatment of cells that had been starved for 6hr with 10⁻⁴M cAMP for as short a time as 30 min was enough to induce 8% of the cells to form spores.
The effects on cAMP-induced differentiation of chemicals that are known to influence development of the wild type were also examined. Both NH₄Cl and KCl inhibited cAMP-induced stalk formation, but had no effect on spore formation. In the presence of arginine, spore formation was induced at a lower concentration of cAMP with higher efficiency. CaCl₂, LiCl and KF had no effect on cAMP-induced differentiation.     

Development of prespore vacuoles inDictyostelium cells differentiating in a liquid shake culture

Summary When shaken in a glucose-albumin-cyclic AMP medium, dissociated aggregative cells form small clumps in which prespore cells differentiate fairly synchronously (Okamoto 1981). Formation of prespore vacuoles (PSVs) in differentiating prespore cells was examined in these culture conditions, by electronmicroscopy and immunocytochemistry.After 6 hours of culture, a typical Golgi apparatus composed of vesicles and stacked flat cisternae develops near the nucleus. FITC-conjugated antispore serum stains a crescent-shaped region in the cells which seems to correspond to the Golgi area. After 9 hours, flat sacs which contain electron dense lining membrane similar to that of PSVs appear alongside Golgi cisternae. Later, partially and fully round PSVs are observed in this region, suggesting that flat sacs round up to become mature PSVs. After 12 hours, as mature PSVs increase in number, they become dispersed throughout the cytoplasm and a typical Golgi apparatus with cisternae disappears. When cultured in a medium devoid of cyclic AMP, cells develop neither Golgi cisternae nor PSVs. These results strongly suggest that PSVs form from Golgi cisternae.     

Separation of Dictyostelium discoideum cells into density classes throughout their development and their relationship to the later cell types

This paper describes a fast, non-destructive method for the separation of large quantities of Dictyostelium discoideum cells into density classes at all stages of development. The cells were separated by low-speed centrifugation on preformed, linear Percoll density gradients. On these gradients, cells at all developmental stages showed a unimodal variation in density and this variation in density rapidly increased during the first hours of development. The density was affected by the amount of salt present in the gradient medium, which suggests that it is regulated by a permeability property of the cells. Slug cells showed a unimodal variation in density and did not form bands corresponding to the cell types. However, were able to isolate density fractions which showed a good enrichment of prespore and prestalk cells: 95% and 90%, respectively. Preaggregation cells separated on density gradients yielded fractions which contained different amounts of three developmentally regulated enzymes. Hence, cells at this stage are already heterogeneous in their enzymatic content. Sorting experiments showed a strong correlation between density and developmental fate; the least dense (light) cells preferentially became prestalk cells, and the dense (heavy) cells became prespore cells. This was found for cells at all developmental stages; even vegetative-stage cells showed considerable heterogeneity with regard to density, which was related to their developmental fate. The light cells become prestalk cells, and the heavy cells become prespore cells. Vegetative cells from the various density fractions differed in their DNA content and temporal onset of mitotic activity when resuspended in medium. Therefore, we suggest that the separation of vegetative cells on density gradients results in a separation of cells into cell-cycle phases. Hence, there appear to be cell-cycle-linked differences among vegetative cells, which bias their differentiation towards either the spore or stalk pathway.     

Cell-type Conversion at Low Temperature in Dictyostelium discoideum

The effects of low temperature (5°C) on cell-type conversion in whole slugs of Dictyostelium discoideum and their anterior prestalk- and posterior prespore-isolates were examined immunohistochemically and electronmicroscopically. When slugs were incubated for nine days at 5°C, the proportion of cells containing spore-antigens increased from about 75% to 85%. More important, the proportion of prespore and spore cells increased from about 3% to 40% in anterior prestalk isolates incubated at 5°C for 12 days, but no cell-type conversion from prespore to prestalk cells occurred in posterior prespore isolates. Therefore, the mechanism regulating the proportions of cells that operates at 21 °C does not operate at low temperature. The cells with full competence for stalk differentiation could change into stalk cells even at 5°C, because a short stalk was always formed when early culminants were transferred to low temperature. The effects of low temperature on several sequential steps of cell differentiation are discussed on the basis of these findings. The ultrastructural characteristics during the process of cell-type conversion are also described.     

Prestalk/prespore differentiation tendency of Dictyostelium discoideum cells as detected by a stalk-specific monoclonal antibody

By the use of a prestalk- and stalk-specific monoclonal antibody, production of prestalk antigen was examined with non-glucose grown [G(-)] and glucose grown [G(+)] cells of Dictyostelium discoideum AX2. Unlike wild type (NC4), some growth phase cells of AX2 were reactive with the antibody. However, G(-) cells contained much more antigen than G(+) cells and the difference between the two remained during the preaggregation period. Besides glucose, the addition of metabolizable, but not nonmetabolizable sugars to both growth phase and preaggregation cells suppressed the production of the prestalk antigen on the one hand and stimulated the accumulation of glycogen on the other hand. When mixed, G(-) cells which produced more prestalk antigen during the preaggregation period remained prestalk cells after aggregation, while G(+) cells which produced less antigen were converted to prespore cells. G(+) cells collected at the stationary phase [G(+)st] were stronger in prestalk sorting tendency than G(+) cells but weaker than G(-) cells. The prestalk antigen content of G(+)st cells prior to aggregation was an intermediate between those of G(-) and G(+) cells. These lead to the conclusion that the prestalk antigen content of preaggregation cells reflect the tendency of the cells toward either prestalk or prespore differentiation after aggregation.     

Bacillus subtilis Cells Lacking Penicillin-Binding Protein 1 Require Increased Levels of Divalent Cations for Growth

Bacillus subtilis strains lacking penicillin-binding protein 1 (PBP1), encoded by ponA, required greater amounts of Mg²⁺ or Ca²⁺ for vegetative growth or spore outgrowth than the wild-type strain and strains lacking other high-molecular-weight (HMW) PBPs. Growth of ponA cells in a medium low in Mg²⁺ also resulted in greatly increased cell bending compared to wild-type cells or cells lacking other HMW PBPs. The addition of high levels of Mg²⁺ to growth media eliminated these phenotypes of a ponA mutant. In contrast to the effects of divalent cations, NaCl did not restore ponA cell growth in a divalent-cation-deficient medium. Surprisingly, wild-type cells swelled and then lysed during both vegetative growth and spore outgrowth when 500 mM NaCl was included in a divalent-cation-deficient medium. Again, Mg²⁺ addition was sufficient to allow normal vegetative growth and spore outgrowth of both wild-type and ponA cells in a medium with 500 mM NaCl. These studies demonstrate that (i) while HMW PBPs possess largely redundant functions in rich medium, when divalent cations are limiting, PBP1 is required for cell growth and spore outgrowth; and (ii) high levels of NaCl induce cell lysis in media deficient in divalent cations during both vegetative growth and spore outgrowth.