Alterations in trehalase solubility during development in the cellular slime mould Dictyostelium discoideum

Previous studies have indicated that during development in the slime mould Dictyostelium discoideum, compartmentation of the isoenzymes of trehalase (alpha, alpha'-trehalose 1-D-glucohydrolase, (EC 3.2.1.28) occurs between the extracellular and intracellular environments. The compartmentation of trehalase between soluble and particulate cell fractions was examined in this work. The trehalase present in crude homogenates prepared during the first 12 h of development was completely soluble. Starting at about the pseudoplasmodial stage (i.e. the 14th hour of development), trehalase activity became associated with insoluble cellular material and this increased to a maximal value in homogenates from mature sorocarps, where 50% of the activity was insoluble. Spore cells accounted for only 2 to 3% of the trehalase associated with mature sorocarps, with the remaining 97% being localized in stalk cell material. Although trehalase recovered from spores was completely soluble, more than half of that from the stalk was recovered in the buffer-insoluble pellet fraction.     

Regulation of trehalase activity during the cell cycle of Saccharomyces cerevisiae

Synchronous cultures of Saccharomyces cerevisiae prepared by selection of small unbudded cells from an elutriating rotor were used to measure trehalase activity during the cell cycle. After the small cells had been removed from the rotor, the remainder was used to prepare asynchronous control cultures. Both synchronous and control cultures were studied for two cell cycles. In asynchronous cultures the trehalase activity of crude cell lysates rose continuously. In synchronized populations trehalase activity increased from the beginning of budding onwards. However, around the period of cell division the enzyme activity dropped rapidly but transiently by more than 5-fold. The same changes were found during the second budding cycle. Measurements of invertase and glucose-6-phosphate dehydrogenase activities in the same synchronous and asynchronous cultures revealed a continuous increase for both enzymes. Incubation of cell lysates with cAMP-dependent protein kinase before assaying for trehalase resulted in a 2-fold enhancement of enzyme activity in asynchronous control cultures. In synchronized cells this treatment also led to a significant stimulation of trehalase activity, and largely abolished the cell-cycle-dependent oscillatory pattern of enzyme activity. These results suggest that the activity of trehalase during the cell cycle is regulated, presumably at the post-translational level, by a phosphorylation-dephosphorylation mechanism.     

Glycogen degradation during migration of presumptive cell types in Dictyostelium discoideum.

During the time course of differentiation in Dictyostelium discoideum, glycogen was found to accumulate from the amoebae stage to the culmination stage of development. Upon sorocarp formation (23 h), glycogen was rapidly degraded. Ultramicrotechniques, utilizing amplification of glycogen by enzymatic cycling, were used to follow glycogen metabolism in pre-stalk and prespore cells during the differentiation cycle. Both cell types accumulated glycogen at nearly the same rate. By the pseudoplasmodium stage of development glycogen had accumulated to 50% of its maximum value, and no differences were found between pre-stalk and pre-spore cells. Glycogen was degraded as pre-stalk cells migrated into the position for stalk construction. At the culmination stage of development stalk cells near the base were devoid of glycogen while pre-stalk cells near the apex of the stalk showed no loss of glycogen. The complete loss of glycogen from stalk cells occurred over a distance occupied by approximately 100 cells, and over a time period of approx. 1 h. Pre-spore cells at the culmination stage showed no loss of glycogen even though separated from stalk cells by only a thin cellulose sheath. The degradation of prespore cell glycogen did not commence until stalk construction was completed and the pre-spore mass had reached the apex of the stalk. Pre-spore cells at the culmination stage contained high levels of glycogen while only 2 h later, total degradation had occurred.     

The calcium content of the cellular slime mold, Dictyostelium discoideum, during development and differentiation

A high calcium concentration is known to induce stalk differentiation of the cellular slime mold D. discoideum. Therefore, the change in the calcium content of this organism during differentiation was studied and found to vary during development, more calcium being found in the anterior prestalk cells of the pseudoplasmodium (slug) than in the posterior prespore cells. It is concluded from the results that calcium is of importance in the cell differentiation of this organism and particularly in stalk formation.     

Evidence that the fertilization envelope blocks sperm entry in eggs of Xenopus laevis: interaction of sperm with isolated envelopes.

Single-celled myxamoebae undergo differentiation into either stalk cells or spore cells during a 24-hr period in Dictyostelium discoideum. This study employed ultramicrochemical techniques and enzymatic cycling to assess the presence of cell-specific events in spore and stalk cells. Freeze-dried sections of one organism were assayed in 0.1 μl of reaction mixture. This method was used to determine the extent of localization of trehalose in spore cells and stalk cells during development.Trehalose was low in the early stages of differentiation to about 20 hr when the level started to increase. In developing spore cells, the trehalose level increased sixfold during the last 5 hr of development. Likewise, the entire stalk contained trehalose when the stalk was first formed. At mature sorocarp, trehalose levels were the same in spores and the apex of the stalk. There was a decreasing gradient of trehalose down the stalk. The bottom one-fourth of the stalk was devoid of this disaccharide. Therefore, trehalose was degraded from an area of the stalk where it was localized earlier in development.The results of this investigation negate the assumption that trehalose is never present in the stalk. Although trehalose was found in spore cells, prestalk cells also contained high trehalose levels. The stalk cell-specific trehalose was not retained during differentiation, however, but was apparently degraded in the mature stalk cell.     

Biological activities of novel derivatives of DIF-1 isolated from Dictyostelium

The differentiation-inducing factor-1 (DIF-1) is a lipophilic signal molecule (chlorinated alkylphenone) that induces stalk cell differentiation in the cellular slime mold Dictyostelium discoideum. In addition, DIF-1 and its derivatives have been shown to possess anti-leukemic activity and glucose consumption-promoting activity in vitro in mammalian cells. In this study, to assess the chemical structure-effect relationship of DIF-1, we synthesized eight derivatives of DIF-1 and investigated their stalk cell-inducing activity in Dictyostelium cells and pharmacological activities in mammalian cells. Of the derivatives, two amide derivatives of DIF-1, whose hydrophobic indexes are close to that of DIF-1, induced stalk cell differentiation as strongly as DIF-1 in Dictyostelium cells. It was also found that some derivatives suppressed cell growth in human K562 leukemia cells and promoted glucose consumption in mouse 3T3-L1 cells. These results give us valuable information as to the chemical structure-effect relationship of DIF-1.     

Changes in activities of several enzymes involved in carbohydrate metabolism during the cell cycle of Saccharomyces cerevisiae.

Activity changes of a number of enzymes involved in carbohydrate metabolism were determined in cell extracts of fractionated exponential-phase populations of Saccharomyces cerevisiae grown under excess glucose. Cell-size fractionation was achieved by an improved centrifugal elutriation procedure. Evidence that the yeast populations had been fractionated according to age in the cell cycle was obtained by examining the various cell fractions for their volume distribution and their microscopic appearance and by flow cytometric analysis of the distribution patterns of cellular DNA and protein contents. Trehalase, hexokinase, pyruvate kinase, phosphofructokinase 1, and fructose-1,6-diphosphatase showed changes in specific activities throughout the cell cycle, whereas the specific activities of alcohol dehydrogenase and glucose-6-phosphate dehydrogenase remained constant. The basal trehalase activity increased substantially (about 20-fold) with bud emergence and decreased again in binucleated cells. However, when the enzyme was activated by pretreatment of the cell extracts with cyclic AMP-dependent protein kinase, no significant fluctuations in activity were seen. These observations strongly favor posttranslational modification through phosphorylation-dephosphorylation as the mechanism underlying the periodic changes in trehalase activity during the cell cycle. As observed for trehalase, the specific activities of hexokinase and phosphofructokinase 1 rose from the beginning of bud formation onward, finally leading to more than eightfold higher values at the end of the S phase. Subsequently, the enzyme activities dropped markedly at later stages of the cycle. Pyruvate kinase activity was relatively low during the G1 phase and the S phase, but increased dramatically (more than 50-fold) during G2. In contrast to the three glycolytic enzymes investigated, the highest specific activity of the gluconeogenic enzyme fructose-1, 6-diphosphatase 1 was found in fractions enriched in either unbudded cells with a single nucleus or binucleated cells. The observed changes in enzyme activities most likely underlie pronounced alterations in carbohydrate metabolism during the cell cycle.     

Induction of stalk cell differentiation by cyclic-AMP in a susceptible variant of Dictyostelium discoideum.

The P-4 variant of Dictyostelium discoideum (DdH) was found to produce a great excess of stalk cells compared to the wild type DdH. If the vegetative cells of P-4 were repeatedly washed, the variant changed back to the wild type phenotype, and if cyclic-AMP was added to the washed P-4 cells, the variant character was restored. Furthermore, if the concentration of added cyclic-AMP was increased, it was possible to induce 100% stalk cells in P-4. Phosphodiesterase would cause the variant to change to the wild type, while 5-AMP and cyclic-nucleotides other than cyclic-AMP have no effect at all. Therefore it was concluded that cyclic-AMP plays a key role in stalk cell differentiation.A comparison between wild type DdH and the variant P-4 showed that DdH is ten times less sensitive to cyclic-AMP induction. They both produce the same amount of cyclic-AMP and extracellular phosphodiesterase, but the specific activity of P-4 cell-bound phosphodiesterase during development is significantly less than that in the DdH. One hypothesis that accounts for the P-4-DdH difference is that because of the lack of cell-bound phosphodiesterase, more cyclic-AMP enters the variant cells and hence more stalk cell differentiation.     

Localization of glycogen phosphorylase in specific cell types during differentiation of Dictyostelium discoideum

The localization of glycogen phosphorylase was studied during the differentiation of prespore and prestalk cells in Dictyostelium discoideum. Ultramicrotechniques were utilized to assay the enzyme activity in cell samples as small as 0.02 μg dry wt in reaction volumes of 0.1 μl. The activity was assayed using an amplification procedure employing the enzymatic cycling of pyridine nucleotides. Glycogen phosphorylase from individual organisms was assayed during the developmental period. Early in development, activity was low but gradually increased to a maximum value at culmination. From culmination to sorocarp, enzyme activity decreased rapidly. Cell-specific assays of spores showed that phosphorylase activity increased slightly to culmination, and then decreased. Prestalk cells showed the greatest activity in the area of stalk sheath construction and elongation. Stalk cells showed a decreasing gradient of enzyme activity from the tip of the stalk to the base. Enzyme activity in the spores may be sufficient to provide glucose units for trehalose synthesis and spore coat production. The prestalk enzyme may degrade glycogen to provide glycosyl units for production of the stalk sheath and trehalose. Possible models of cell-specific biochemical events in Dictyostelium discoideum are discussed.     

Cell cycle phase in Dictyostelium discoideum is correlated with the expression of cyclic AMP production, detection, and degradation. Involvement of cyclic AMP signaling in cell sorting

Cell cycle phase in Dictyostelium is correlated with a different preference for either spore or stalk differentiation. Cells which start development early in the cell cycle (E cells) exhibit a strong tendency to sort to the prestalk region of slugs, while late cell cycle cells (L cells) sort to the prespore region. We investigated the expression of the cAMP chemotactic system during development of synchronized E and L cells and found that E cells exhibit cAMP-binding activity, cell surface cAMP-phosphodiesterase (mPDE) activity, and the ability to relay cAMP signals at least 2 hr earlier and to higher levels than L cells. We hypothesize that E cells are prestalk sorters because they are the first to initiate aggregation centers and respond most effectively with chemotaxis and signal relay.     

A constitutive, heat shock-activated neutral trehalase occurs in Schizosaccharomyces pombe in addition to the sporulation-specific acid trehalase

Trehalase was studied in Schizosaccharomyces pombe cells growing vegetatively on minimal medium and in sporulating cultures. Acid trehalase activity, measured at pH 4.2, was absent in vegetative cells and occurred only in asci, indicating that this activity represented the sporulation-specific trehalase reported previously. In contrast, neutral trehalase, measured at pH 6.0, was constitutively present in vegetative cells during the exponential and stationary growth phase as well as in asci. In vegetative cells, neutral trehalase did not sediment with cell walls, suggesting a cytoplasmic localization. Its activity increased ten-fold when growing cells were subjected to heat treatment of 2 h. Neutral trehalase from heat-treated cells had a pH optimum of 6.0 and was almost completely inhibited by 3 mM ZnCl2. Acid trehalase activity could be measured in intact asci, indicating that it is localized in the ascus cell walls, while neutral trehalase was not detectable in intact asci and appeared to be present primarily in the walls of ascospores and in the ascus epiplasm.     

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.     

Changes in enzyme activity during differentiation in Chlamydomonas reinhardtii

The patterns of alanine dehydrogenase, glutamate dehydrogenase and malate dehydrogenase activity were studied during the normal vegetative cell cycle and during the process of gametic differentiation and dedifferentiation in synchronized cultures of Chlamydomonas reinhardtii. During all three phases of growth and differentiation the synthesis of DNA was also measured. During gametic differentiation all three enzyme levels were suppressed compared to vegetative cells although DNA and cell number were comparable. During gametic dedifferentiation no DNA synthesis occurred during the first 24 h cycle and only a doubling during the second. It was not until the third cycle that a normal 4-fold increase in DNA was observed. Cell number followed a similar pattern. Athough the levels of alanine dehydrogenase and malate dehydrogenase were uniformly low during the first cycle when glutamate dehydrogenase increased 4-fold, during the second cycle the patterns of these enzymes changed markedly. The enzymes did not attain levels characteristic of vegetative cells until the third cycle.     

ATP, trehalose, glucose and ammonium ion localization in the two cell types of Dictyostelium discoideum

Ultra-microfluorometric techniques were adapted to follow several compounds related to energy metabolism through the developmental cycle of Dictyostelium discoideum. Each compound (ATP, trehalose, glucose, and ammonium ion) was found to be present in stalk and/or spore cells. The accumulation of NH4+ was interpreted as an indication of protein degradation, a source of energy in this organism. During the early stages of differentiation NH4+ was localized only in prestalk cells. However, it accumulated in spore cells during culmination such that levels were comparable in the two cell types by the end of development. Trehalose, an energy source for germinating spores, was found in both cell types but was preferentially degraded in stalk cells late in development. Glucose, the degradation product of trehalose, was localized in prestalk cells and varied inversely with trehalose levels. ATP was not localized in a specific cell type during development. However, ATP declined in stalk cells at an earlier stage of development.     

Cell specific activity of trehalose-6-phosphate synthetase during differentiation of Dictyostelium discoideum.

Trehalose-6-P synthetase activity was low at the beginning of the life cycle of Dictyostelium discoideum, reached maximum activity at 20 h, and decreased at late sorocarp. Enzyme activity in developing spore cells increased 10-fold during differentiation from myxamoebae (0 h) to the culmination stage (20 h) and decreased slightly at sorocarp (24 h). Activity was similar in spore cells at the apex of the stalk. The activities in the stalk cells were dependent upon their position in the developing stalk. There was a decreasing gradient of activity from the apex to the base of the stalk.     

A genetic melanotic neoplasm of Drosophila melanogaster

The construction of mature fruiting bodies occurs during the culmination stage of development of Dictyostelium discoideum. These contain at least two different cell types, spores and stalks, which originate from an initially homogenous population of vegetative amoebas. As an attempt to identify proteins whose synthesis is regulated in each cell type during differentiation, we have analyzed the two-dimensional profiles of proteins synthesized by spore and stalk cells during the culmination stage. We have identified 5 major polypeptides which are specifically synthesized by spore cells during culmination and 9 which are only made by stalk cells. Furthermore, synthesis of about 20 polypeptides appears to be enriched either in the spore or in the stalk cells. We also show that synthesis of actin, a major protein synthesized during Dictyostelium development, is specifically inhibited in the spore cells during culmination. Synthesis of most of the cell type-specific proteins initiates at 19–20 hr, during culmination. Moreover, the proteins whose synthesis is induced after formation of tight aggregates, the time when the major change in gene expression occurs, are not specifically incorporated into spores or stalk cells, and appear to be synthesized by both cell types. We conclude that a new class of genes is expressed during the culmination stage in Dictyostelium, giving rise to specific patterns of protein synthesis in spore and stalk cells.     

Differentiation capabilities of mouse optic stalk in isolation of its immediate in vivo environment

During normal in vivo development, the optic stalk gives rise only to macroglial cells. When we cultured optic stalks isolated from their immediate in situ environment, we found that optic stalks obtained from embryos at Theiler stages 16 to 19 gave rise to both neurons and glial precursor cells, whereas optic stalks obtained from embryos at stages 20 to 23 gave rise to only glial precursor cells. Between stages 19 and 20 (a period of 12 hr of development) the optic stalk changes from a pseudostratified to a simple epithelium, and concomitant with these changes is the growth of the neural retinal axons along the optic stalk. An attractive hypothesis to explain these observations is that the environmental cues that restrict the differentiation capability of the optic stalk ventricular cell population in vivo emanate from the retinal axons. Whether this is due to a restriction in the differentiation capability of a pleuripotential ventricular cell or to a selective cell death of a subpopulation of ventricular cells already committed to the neuronal lineage of differentiation is not yet resolved.     

Characteristics of trehalase inCandida tropicalis

Summary The trehalase content of different yeasts varies widely. A strain ofCandida tropicalis was found to be the best source of this enzyme among the yeasts tested. The trehalase activity in this yeast could be increased 8.5 times by growing it on trehalose rather than glucose. Thus trehalase is an adaptive enzyme inC. tropicalis. It was found that the amount of trehalase which could be solubilized increased with increasing pH during autolysis of the cells, none being released from the cell debris at pH 4.5 and most at pH 6.3. Some evidence was obtained to show that the solubilization was caused by an enzyme. The stability of trehalase under various conditions was studied. A partial purification was achieved by precipitation with 40% ethanol at a temperature of −18°C. The maximum temperature of the enzyme was 48°C., and the optimum pH ranged from 4.1 to 5.3     

Differential Extraction of N-Acetylglucosaminidase and Trehalase from the Cell Envelope of Candida albicans

Molloy, C., Shepherd, M. G., and Sullivan, P. A. 1995. Differential extraction of N-acetylglucosaminidase and trehalase from the cell envelope of Candida albicans. Experimental Mycology 19, 178-185. Dithiothreitol (DTT) extraction of N-acetylglucosaminidase and trehalase from intact Candida albicans ATCC 10261 cells was monitored as an index of cell envelope porosity during N-acetylglucosamine-induced morphogenesis. Trehalase, which is secreted into the cell envelope during starvation and bud-formation, displayed similar extraction kinetics in starved, germ tube-forming, and bud-forming cells, indicating that the mother cell wall remains largely unchanged during morphogenic outgrowth and that the porosity of bud and mother cell walls is similar. N-acetylglucosaminidase, which is secreted specifically during morphogenesis, was released eightfold more rapidly from germ tube-forming than bud-forming cells, reflecting major differences in porosity between bud and germ tube. In addition, by assaying DTT extracts and extracted cell residues, it was found that the total extracellular N -acetylglucosaminidase activity increased 2- to 2.5-fold during DTT treatment. Thus, DTT unmasks a cryptic form of N-acetylglucosaminidase. The cryptic activity was associated with the cell wall fraction.     

Ultrastructural and lectin binding changes during the formation of the animal dimple in oocytes of Discoglossus pictus (Anura)

The numbers of spores, stalk cells, and basal disk cells in fruiting bodies of Dictyostelium discoideum were estimated by direct cell counting. It was found that the ratios of differentiated cells varied with the number of cells in the fruiting body. Hence, this invalidates, in D. discoideum at least, an assumption used in many theories of differentiation that proportions do not vary with size. Simple statistical analysis showed that a semilogarithmic equation could describe the relationship of spore to stalk cell number and spore to basal disk cell number, whereas a double-logarithmic equation described the basal disk and stalk cell number relationship. Studies under different environmental conditions and with different strains suggest that the basic equations describing the relationships are conserved. However, quantitative differences in the proportioning of the cell types have been observed. Previous papers concerning the proportions of D. discoideum are reviewed, and the implications of the results, in regard to theories of differentiation, are analyzed.