An immunological assessment of lysosomal enzymes and other macromolecules sulfated during vegetative growth of Dictyostelium discoideum

Western blotting and immunoprecipitation data indicated that lysosomal enzymes represent a subset of the sulfated macromolecules present in vegetative Dictyostelium discoideum amoebae and account for less than 2.5% of the total sulfate incorporated during vegetative growth. These data suggest that the majority of the highly sulfated macromolecules of vegetative D. discoideum amoebae are not related to the lysosomal enzymes.     

Oligosaccharide structure determination of glycoconjugates using lectins

Lectins, the divalent or polyvalent (glyco) proteins of non-immune origin of the cells agglutinate cells or other materials, that display more than one saccharide of sufficient complementarity. Lectins considered ‘identical’ in terms of mono-and disaccharide specificity can be differentiated by their ability to recognise the fine differences in more complex structures. The present review discusses the interaction of lectins with various oligosaccharides and their resultant separations due to structural variations.     

The glycoproteins of Dictyostelium discoideum. Changes during development.

The glycoproteins of D. discoideum have been analyzed by direct binding of radio-iodinated lectins to SDS gels of the successive developmental stages. Compared with the total pattern of proteins, many changes are found in the glycoproteins during development. WGA reacts with few gel bands from the vegetative cells and most of these, including a very intense band at the top of the gel, are lost during the first few hours of development. Approximately half-way through the developmental cycle at least 14 new glycoproteins reacting with WGA begin to appear and progressively accumulate. In contrast, ConA labels many glycoproteins over the complete molecular weight range and most are unaffected during development. Lectins which bind fucose label a single component at the top of the gel of vegetative cells and this decreases rapidly as development begins. No other reactive gel bands are revealed by fucose-binding lectins until the final stages of spore and stalk formation, when four high molecular weight glycoproteins are detected. Lectins specific for terminal galactose residues and for N-acetyl-galactosamine, including the intrinsic lectins produced by D. discoideum during its development, failed to reveal any reactive glycoproteins.     

Effects of pteridines on the filopodia of Dictyostelium discoideum vegetative amoebae

Living vegetative amoebae of NC-4H Dictyostelium discoideum were studied to determine if a variety of pteridines had any effect on the filopodia. We observed that production, elongation, and branching of these filopodia were stimulated by pteridines that are chemoattractants for cells of this strain. This stimulation occurs at chemotactically effective concentrations and is observed before motility is evident. A relationship between filopodia and chemoattractant signal processing is discussed.     

Differential in vitro activation and deactivation of cysteine proteinases isolated during spore germination and vegetative growth of Dictyostelium discoideum.

Acid-activatable cysteine proteinases of Dictyostelium discoideum were first identified in spore extracts of strain SG1 using gelatin/SDS/PAGE, followed by acid treatments. Here we utilized the technique of acid activation to identify cryptic cysteine proteinases throughout auto-induced and heat-induced spore germination of D. discoideum strain SG2 and SG1. The major acid-activatable cysteine proteinase identified in SG2 and SG1 spore extracts was ddCP38 (D. discoideum cysteine proteinase with a molecular mass of 38 kDa) and ddCP48, respectively. Further investigation of these enzymes revealed that they were also base deactivatable with a treatment of ammonium chloride directly following acid activation. However, the most intriguing observation was the reversibility of the effects of base deactivation on the enzymes following a second treatment with acetic acid. Thus, we hypothesize that, unlike most mammalian cysteine proteinases which generally require the cleavage of a pro-peptide region for activation, these cysteine proteinases of D. discoideum likely undergo reversible conformational changes between latent and active forms. Moreover, we were able to detect these cryptic cysteine proteinases in the vegetative cells and early aggregates of both strains SG1 and SG2. Studies using 4-[(2S, 3S)-3-carboxyoxiran-2-ylcarbonyl-L-leucylamido]buty lguanidine, a cysteine proteinase inhibitor, revealed that acid activation of a portion of these proteinases was still achievable even after incubation with the inhibitor, further supporting the concept of two stable and reversible conformational arrangements of the enzymes. Thus, we speculate that the pH shuffles that modulate proteinase conformation and activity in vitro may be a reflection of the in vivo regulation of these enzymes via H+-ATPases and ammonia.     

The absence of a growth inhibitor during the log phase of growth of Dictyostelium discoideum

When amebae of the cellular slime mold Dictyostelium discoideum Ax-3 reach stationary phase in liquid nutrient medium, an extracellular factor appears which will inhibit cell multiplication when added to a logarithmically multiplying culture (Yarger et al., 1974). Recently, Hanish (1975) presented evidence that an inhibitor also accumulated during log phase and concluded that the cessation of cell multiplication and the final cell concentration were controlled by the accumulation of this inhibitor to an effective level. We have reexamined this extremely interesting observation, but have found no evidence to support Hanish's conclusions.     

Half-lives of messenger RNA species during growth and differentiation of Dictyostelium discoideum

The half-lives of functional messenger RNAs were determined by a method employing the drugs actinomycin D and daunomycin for the inhibition of mRNA synthesis; the activity of extracted mRNAs was determined by an in vitro translation assay. Several controls indicated that this method yielded reliable values for mRNA half-lives; in particular, the declining rate of protein synthesis in the presence of the drugs is due predominantly to the decay of translatable mRNA. This method was used to determine the half-lives of two specific mRNAs—encoding actin and a protein of MW 51,000—as well as that of total cytoplasmic mRNA activity during growth and at several times in differentiation. The half-lives of at least these two mRNAs were shown to be distinctly different from that of the total mRNA population—about 4 hr. However, no significant change in any of these half-lives was observed between growing and developing cells. Therefore wholesale alterations in the degradation rates of total and at least specific messages do not appear to play a role in the regulation of gene expression during Dictyostelium development.     

The effect of respiratory inhibitors during growth and development of Dictyostelium discoideum

Two major pathways of electron transport to oxygen were identified in intact cells of Dictyostelium discoideum, a cyanide-sensitive pathway and a cyanide-insensitive, salicylhydroxamic-acid-sensitive pathway. The extent to which each pathway contributed to the total respiratory activity was shown to change during exponential growth and throughout development. During exponential growth both pathways appear to be utilized to varying degrees dependent on culture age, during late exponential growth the activity of the salicylhydroxamic-acid-sensitive pathway would seem to be almost totally lost. During development the cyanide-sensitive pathway appears to be dominant up to the aggregation stage, but both pathways are active in pseudoplasmodial cells. It is also suggested that the presence of iron in the growth medium may be essential directly or indirectly, for the maintained activity of the salicylhydroxamic-acid-sensitive pathway late in exponential growth.     

Role of cAMP-dependent protein kinase during growth and early development of Dictyostelium discoideum

cAMP-dependent protein kinase (PKA) is an essential regulator of gene expression and cell differentiation during multicellular development of Dictyostelium discoideum. Here we show that PKA activity also regulates gene expression during the growth phase and at the transition from growth to development. Overexpression of PKA leads to overexpression of the discoidinIgamma promoter, while expression of the discoidinIgamma promoter is reduced when PKA activity is reduced, either by expression of a dominant negative mutant of the regulatory subunit or by disruption of the gene for the catalytic subunit (PKA-C). The discoidin phenotype of PKA-C null cells is cell autonomous. In particular, normal secretion of discoidin-inducing factors was demonstrated. In addition, PKA-C null cells are able to respond to media conditioned by PSF and CMF. We conclude that PKA is a major activator of discoidin expression. However, it is not required for production or transduction of the inducing extracellular signals. Therefore, PKA-dependent and PKA-independent pathways regulate the expression of the discoidin genes.     

Changes in the DNA content of amoebae of Dictyostelium discoideum during growth and development.

A fluorimetric assay has been used to determine the DNA content of amoebae of Dictyostelium discoideum during growth and development. Amoebae grown in axenic culture tended to be multinucleate and had a greater DNA content than amoebae grown with a bacterial substrate, which were mononucleate. During the first 10 h of development there was little change in the DNA content of amoebae grown with a bacterial substrate, but the average DNA content per cell in amoebae grown axenically decreased as the amoebae became virtually mononucleate. Amoebae at 10 h development that had been harvested during exponential axenic growth were divided into two populations by countercurrent distribution in a polymer two-phase system. DNA content indicated that one population was largely in the G2-phase of the cell cycle, whereas the other population was largely in the G1-phase. Similar results were obtained at 10 h development with amoebae harvested during the stationary phase of axenic growth, although these amoebae start development all in the G2-phase of the cell cycle. Spores had a low DNA content, indicating that they were in G1-phase. It is proposed that all amoebae in G2-phase after early development differentiate, after mitosis, into spores and that stalk cells are formed from amoebae that remain in G1-phase after 10 h development.     

The expression of glycoproteins in the extracellular matrix of the cellular slime mold Dictyostelium discoideum

In this report we examine the accumulation of glycoconjugates in the extracellular medium and insoluble matrices surrounding developing cells of the cellular slime mold Dictyostelium discoideum. Conditions were employed which permitted advanced development (slug stage and beyond) in suspension culture. Under these conditions, up to one-third of the total culture protein appeared as non-sedimentable, extracellular material over the course of 48 h of incubation. Most of the secreted molecules expressed carbohydrate antigens (glycoantigens) as detected by Western blotting, using a panel of six monoclonal antibodies. Since the glycoantigens are secreted, immunoelectron microscopy was used to localize the glycoantigens in the extracellular matrices surrounding normally developing cells, including the slime sheath, stalk tube, inner spore coat, outer spore coat, and intercellular fluid between spores. Each glycoantigen had a characteristic distribution, and each extracellular matrix space contained a unique combination of glycoantigens. Thus, although each of these matrices (except inter-spore fluid) contains cellulose as a primary component, they could be distinguished on the basis of their glycoantigen and, by inference, glycoprotein compositions. Furthermore, there were differences between anterior and posterior regions of both slime sheaths and stalk tubes. These observations show that secretion as detected in suspension culture occurs under normal conditions as a part of the process of depositing extracellular matrices around the cells. The distributions show that the cell aggregate positionally regulates the expression and deposition of secretory glycoproteins; the resultant patterns of expression of unique protein-linked carbohydrate structures imply a functional role in matrix organization and possibly cell activity which can now be explored.     

盘基网柄菌(Dictyostelium discoideum)吞噬作用中肌动蛋白多聚化及其调节

肌动蛋白是盘基网柄菌(Dictyostelium discoideum)细胞吞噬过程中的关键组分,通过其细胞内的定位和多聚化形式在确定的时间和地点连接特定的分子,使吞噬过程得以完成。profilin是肌动蛋白多聚化的重要调节分子,在磷脂酰肌醇信号转导与细胞骨架相交处起关键作用。许多小分子G蛋白参与细胞骨架调节,CAP蛋白是两者间重要连接分子。所以,吞噬作用是细胞内诸分子协同作用的结果。     

A labile acid phosphatase isozyme associated with the surface of vegetative Dictyostelium discoideum cells.

A minor acid phosphatase isozyme (acid phosphatase I) of vegetative Dictyostelium discoideum amebae has been shown to be associated exclusively with the external surface of the plasma membrane. The isozyme is not present in phagocytic vacuoles isolated with latex beads. The isozyme disappears from cells removed from nutrient medium and does not reappear during differentiation. When inhibitors of protein synthesis (e.g. cycloheximide, chloral hydrate, concanavalin A) are added to cells growing in nutrient medium, acid phosphatase I is rapidly lost. It appears that the level of protein synthesis need only be moderately reduced (less than 25%) to induce loss of enzyme activity. Treatment with inhibitors of DNA and RNA synthesis for up to 2 h had no effect on isozyme activity. It is postulated that the cells are able to "sense" (through the reduction in levels of protein synthesis) when external conditions become unfavorable, and immediately respond by reducing the activity of enzymes involved in maintaining contact with the extracellular environment. The closed system thought necessary for differentiation would then be created.     

Products of endogenous N-acetylglucosaminyltransferase activity of Dictyostelium discoideum during growth and early development.

In the presence of Mn2+ and uridine diphosphate-N-acetyl-D-[14C]glucosamine, a total particulate fraction prepared from Dictyostelium discoideum amoebae catalyzed the transfer of N-acetyl[14C]glucosamine to endogenous membrane protein acceptors. No transfer to lipid acceptors was evident. The 14C products obtained from growth-phase and aggregation-competent amoebae were converted to glycopeptides by pronase digestion. The respective glycopeptides appeared identical in their chemical and chromatographic properties, suggesting that the same activity was functioning in both growing and differentiating cells. The results provided no evidence for developmental regulation of this activity in D. discoideum.     

Cell-cycle progression during the development of Dictyostelium discoideum and its relation to the subsequent cell-sorting in the multicellular structures

Previous studies have shown that the cell-cycle phase at the onset of starvation is a naturally occurring variable that is closely involved in the subsequent sorting and differentiation of cells during Dictyostelium development. Here the cell-cycle progression during the development of D. discoideum Ax-2 cells and its relation to the subsequent cell-sorting were analyzed in detail using synchronized cells and their pulse-labeling by 5'-bromodeoxyuridine (BrdU). Measurements of cell number and nuclearity provided evidence that about 80% of cells progressed their cell-cycle after formation of multicellular structures (mounds). Many cells (T7 cells) starved at mid–late G2-phase (just before the PS-point from which cells initiate development when starved) progressed to the cell-cycle after mound formation. In contrast, a less amount of cells (T1 cells) starved at late G2-phase (just after the PS-point) progressed through the cell-cycle after mound formation. The significance of cell-cycle progression presented here is discussed, with reference to cell differentiation and pattern formation.     

Regulation and secretion of early developmentally controlled enzymes during axenic growth in Dictyostelium discoideum

The four earliest developmentally controlled enzymes in the cellular slime mold, Dictyostelium discoideum, accumulate during axenic growth in rich media. We have shown that at low cell titers the specific activities of N-acetylglucosaminidase, α-mannosidase, leucine aminopeptidase, and alanine transaminase are each at very low or vegetative levels comparable to amoebae which have been grown on bacteria as the food source. During the exponential phase of growth all four enzymes accumulate dramatically reaching cellular specific activities at least as high as during development. The magnitude of this accumulation is influenced by alterations in the growth medium. We suggest that these results, combined with those of prior investigations, indicate that a restricted segment of early development is initiated during axenic growth. This means that growth and early development are not mutually exclusive events in this organism. The secretion of lysosomal enzymes is also affected by the composition of the growth media. In all media, including growth in bacterial suspensions, lysosomal enzymes are secreted in significant quantities. There is a correspondence in the effects of media composition on the secretion of these enzymes and on the regulation of developmentally controlled enzymes during axenic growth. The secretion of lysosomal enzymes that are not developmentally regulated is affected in these media, suggesting that the regulation and secretion of these enzymes are under separate control. It is clear that studies of the regulation of lysosomal enzymes in this organism must take into account the secretion of the enzymes as well as their cellular specific activities to properly reflect levels of gene expression.