Metabolism of the cellular slime mould Dictyostelium discoideum grown in axenic culture

1. The DNA, RNA, protein and carbohydrate contents of myxamoebae of Dictyostelium discoideum strain Ax-2 were measured after growth on bacteria or in various axenic media. 2. Myxamoebae grown in the different axenic media have similar DNA, RNA and protein contents, but there are marked differences in the contents of glycogen and free sugars. The DNA and protein contents of myxamoebae grown on bacteria are different from those in myxamoebae grown axenically. 3. Approximately half the DNA found in myxamoebae grown on bacteria is of bacterial rather than of slime-mould origin. 4. The specific activities of some enzymes (including UDP-glucose pyrophosphorylase) are higher in myxamoebae grown axenically than in myxamoebae grown on bacteria. Nevertheless the characteristic increase in the specific activity of UDP-glucose pyrophosphorylase occurring during differentiation of cells of the wild-type strain NC-4 is also found in cells grown axenically. 5. The rate of amino acid oxidation during axenic growth of the myxamoebae is decreased when the cells are supplied with glucose.     

Glycogen synthetase and the control of glycogen synthesis in the cellular slime mould Dictyostelium discoideum during cell differentiation

1. The variation in cellular glycogen content of differentiating cells derived from myxamoebae that initially contained a wide range of glycogen contents (0.047-5.56mg of glycogen/10(8) myxamoebae) has been studied. 2. Myxamoebae that initially contained 0.047-3.62mg of glycogen/10(8) myxamoebae all gave rise to fruiting bodies that contained similar amounts of glycogen (0.06-0.11mg of glycogen/10(8) cells) but myxamoebae that initially contained 5.56mg of glycogen formed fruiting bodies containing 0.5mg of glycogen/10(8) cells. 3. Despite the high net rate of glycogen disappearance (during cell differentiation) from cells that contained more than 2mg of glycogen/10(8) cells initially, there were still significant variations in the rate of glycogen synthesis. The rate of glycogen synthesis reached a peak at the aggregation stage. 4. Evidence is presented showing that the rate of this synthesis of glycogen is controlled by factors other than the intracellular concentration of glycogen synthetase. 5. Our results are discussed in the context of the theory that the rates of glycogen synthesis and degradation act as a control mechanism for cell differentiation. 6. Criteria are discussed for deciding whether a biochemical event is causally or secondarily related to morphogenesis.     

Quantitative cytophotometric determination of DNA, RNA and lysine bound protein in relationship to zygote formation and protein synthesis in myxamoebae and swarm cells of Didymium iridis

Quantitative cytochemical determinations were made of the DNA of zygotes formed from myxamoebae and swarm cells of Didymium iridis. The nuclear and cytoplasmic RNA and lysine bound protein of these cells were also measured. Significant zygote formation in myxamoebae crosses began at 20-30 min, while swarmers required 35-40 min. Myxamoebae, however, demonstrated a greater ability to form zygotes. The total cytoplasmic RNA and protein bound lysine for myxamoebae was higher than that of the swarmer cells. This observed decrease in swarmers may be due to reduced protein synthesis. Values for nuclear RNA were higher in the myxamoebae, but nuclear lysine bound protein was higher in the swarmers. The data presented suggest that prefusion swarmers, after replicating their DNA, go into a period of G2 arrest and remain in this condition postfusion. In contrast, prefusion myxamoebae readily divide after DNA replication, and continue to synthesize nuclear DNA, and to divide after fusion.     

In vivo crosslinking of nuclear proteins to DNA by cis-diamminedichloroplatinum (II) in differentiating rat myoblasts

When cells are briefly exposed to cis-diamminedichloroplatinum (II) before lysis in high sodium dodecyl sulfate-urea solutions, the high molecular-weight nucleic acids pelleted by ultracentrifugation contain an increased level of bound proteins when compared to a similar fraction from untreated cells. Subsequent shearing of the pelleted DNA followed by treatment with DNase permits electrophoretic and immunoblot analysis of the crosslinked proteins. In the present study such experiments were carried out with reference to nuclear envelope pore complex proteins in the differentiating L8 rat skeletal muscle cells. The results show that (i) whereas the major lamin proteins crosslinked to DNA in both myoblast and myotubes, lamin B is crosslinked to a greater extent to DNA in myotubes; (ii) a 62-kDa lectin-binding glycoprotein is apparently situated differently with respect to DNA in myotube nuclei; and (iii) the crosslinking pattern of the nuclear matrix proteins to DNA is qualitatively similar in myoblast and myotubes. In addition, lamin C', a modified form of lamin C, not observed in intact nonmuscle cells previously [Glass et al. (1985) J. Biol. Chem. 260, 1895-1900], exists as a native component of the nuclear lamina in rat skeletal myotubes but not in myoblasts. These results point to significant structural alterations in the proteins of the nuclear lamina-pore complex during myogenesis.     

Direct transfer of coliphage lambda DNA from Escherichia coli to cellular slime mold Dictyostelium discoideum

Dictyostelium discoideum myxamoebae were cultured with Escherichia coli cells infected with lambda phage in the presence of chloramphenicol. After eliminating the uningested bacteria by repeated centrifugation in a Percoll gradient, we examined the myxamoeba cytoplasm (not the food vacuole) for the presence of phage DNA. A significant amount of DNA extracted from the myxamoebae was hybridizable with purified phage lambda DNA, and capable of forming phage particles when packaged in vitro with phage lambda proteins. The EcoRI restriction maps of the phages recovered from the plaques were identical to that of the infecting phage. These results strongly suggest that phage DNA molecules were taken up by the cellular slime mold cells and that at least some fraction existed in intact form.     

In vivo crosslinking of nuclear proteins to DNA by cis-diamminedichloroplatinum (II) in differentiating rat myoblasts

When cells are briefly exposed to cis-diamminedichloroplatinum (II) before lysis in high sodium dodecyl sulfate-urea solutions, the high molecular-weight nucleic acids pelleted by ultracentrifugation contain an increased level of bound proteins when compared to a similar fraction from untreated cells. Subsequent shearing of the pelleted DNA followed by treatment with DNase permits electrophoretic and immunoblot analysis of the crosslinked proteins. In the present study such experiments were carried out with reference to nuclear envelope pore complex proteins in the differentiating L8 rat skeletal muscle cells. The results show that (i) whereas the major lamin proteins crosslinked to DNA in both myoblast and myotubes, lamin B is crosslinked to a greater extent to DNA in myotubes; (ii) a 62-kDa lectin-binding glycoprotein is apparently situated differently with respect to DNA in myotube nuclei; and (iii) the crosslinking pattern of the nuclear matrix proteins to DNA is qualitatively similar in myoblast and myotubes. In addition, lamin C′, a modified form of lamin C, not observed in intact nonmuscle cells previously [[31.] J. Biol. Chem. 260, 1895–1900], exists as a native component of the nuclear lamina in rat skeletal myotubes but not in myoblasts. These results point to significant structural alterations in the proteins of the nuclear lamina-pore complex during myogenesis.     

Enzyme synthesis in the cellular slime mould Dictyostelium discoideum during the differentiation of myxamoebae grown axenically

1. Myxamoebae of the cellular slime mould Dictyostelium discoideum Ax-2 were grown on different media, and were harvested either in the stationary or exponential phases of the growth cycle to yield samples of myxamoebae differing in enzymic composition. 2. Morphogenesis and cell differentiation phenomena in D. discoideum appear to be similar in myxamoebae grown and harvested under different conditions. 3. The specific activity of the enzymes beta-N-acetylglucosaminidase, acid phosphatase, alpha-mannosidase, beta-glucosidase and alkaline phosphatase have been determined during cell differentiation of myxamoebae grown and harvested under different conditions. 4. The pattern of synthesis of these enzymes, all of which have been claimed to be part of the ;developmental programme', either remains unaffected despite the origin of the myxamoebae (alkaline phosphatase) or is qualitatively similar but quantitatively affected (acid phosphatase, beta-glucosidase) or is both qualitatively and quantitatively affected by changes in the myxamoebae (alpha-mannosidase, beta-N-acetylglucosaminidase). 5. The implications of these results for the concept of a ;developmental programme' are discussed.     

Apogamic induction of haploid plasmodia in a myxomycete,Didymium iridis

Interisolate crosses between haploid (mean DNA = 0.32) CR 5-5 (A²) myxamoebae and polyploid (mean DNA = 1.80) CR 2–25 (A⁵) myxamoebae of the myxomycete Didymium iridis result in plasmodia that have the haploid (mean DNA = 0.32) DNA content rather than the predicted polyploid value. F₁ clones possess the mating type allele of the CR 5-5 clone only, and they also have the same mean DNA content as CR 5-5 myxamoebae. Crosses between these F₁ clones and CR 2–25 myxamoebae again resulted in the production of haploid plasmodia. Hence, the polyploid CR 2–25 clone appears to induce the CR 5-5 clone to produce plasmodia without involving itself in nuclear fusion.     

Alpha-fetoprotein in the brain of developing rats and pigs. An immunofluorescent study of cell-and-tissue differentiation

Alpha-fetoprotein (AFP) and some other serum proteins have been studied in the developing brain of rats and pigs using the indirect immunofluorescence technique. AFP is shown to be present in the ventricular ependyma, meningeal envelopes, the choroid plexus, blood vessel walls and in a wide scale of differentiating parenchymal cells ever since early embryonic ages of both species. In brain parenchyma the content of AFP is low in immature germinative cells; in both species it starts increasing in postmigratory neuroblasts and reaches a peak at the time of accelerated nerve cell differentiation. In rats, the amount of AFP is highest towards the end of the first postnatal week; then it starts decreasing and disappears towards the end of the 3rd week. In both species AFP is localized in the cytoplasm of nerve cell perikarya and their differentiating processes. Higher concentration of this protein has often been observed at the axonal pole of the cytoplasm of differentiating pyramidal neurons. Immunoglobulin G has been found in non-parenchymal structures, and small amounts also in parenchymal cells of embryonic and early postnatal rats following a pattern of cell-and-tissue distribution similar to that of AFP. In pigs, a low amount of albumin has been shown in differentiating leptomeninges. These data suggest uptake of AFP, and some other serum proteins, from the cerebrospinal fluid into cells of the immature rat and pig brain and its increase (or higher binding) in differentiating neurons.     

A bacterial factor induces changes in cysteine proteinase forms in the cellular slime mould Dictyostelium discoideum.

The electrophoretic pattern of cysteine proteinases in axenically grown myxamoebae of Dictyostelium discoideum can be altered by the addition of either Gram-negative (Klebsiella aerogenes, Escherichia coli) or Gram-positive (Micrococcus lysodeikticus, Bacillus subtilis) bacteria to the culture. No changes occurred, however, if either yeast or latex beads were used in place of bacteria. The changes involved the simultaneous loss of proteinases characteristic of the axenic cells (the A-forms) and the acquisition of those found in cells which have been grown on bacteria (the B-forms). Using K. aerogenes the conversion was complete within 4 h. Extracellular proteinase activity was unaffected during this period. After the D. discoideum cells had been lysed, no equivalent change in proteinase band pattern could be produced either by prolonged incubation of cell extracts or by treatment with proteinases. An identical conversion could be induced in cultures of myxamoebae by a factor, cysteine proteinase converting factor (CPCF), present in the 15,000 g supernatant of a sonicated suspension of K. aerogenes. CPCF was macromolecular, as demonstrated by both ultrafiltration and gel filtration, acid-precipitable, but was soluble in ethanol or alkali. Its activity was unaffected by treatment with trypsin. The results suggested that CPCF might be a component of the bacterial cell wall, and since its activity was affected by lysozyme treatment, peptidoglycan is implicated. The results can be interpreted in terms of a novel nutrient-dependent post-translational change which affected most of the cysteine proteinases present in D. discoideum myxamoebae.     

Comparative microphotometric studies of DNA and arginine in plant nuclei

Summary Photometric measurements have been made of the amounts of stain formed in the Feulgen (DNA) and Sakaguchi (arginine) reactions in plant nuclei of differing ploidy.In nuclei of diploid and tetraploid plants of Tradescantia ohioensis and of diploid, triploid and tetraploid plants of Ranunculus ficaria, both Feulgen and Sakaguchi values gave ratios which agreed closely with the ratios of the number of chromatids known to be present. The Feulgen/ Sakaguchi ratio for each of the different types of nuclei measured was very similar both within and between these two species.In the interphase nuclei of five different species, both Feulgen and Sakaguchi values gave bimodal distributions. In the nuclei of differentiating cells, the proportions of values falling into each of the 2C, 4C or 8C classes were the same for both stains.Measurements of the amounts of both stains were made in sequence on the same individual nuclei and a positive correlation found between the two sets of values.In nuclei from differentiating cells of Vicia faba primary roots, the Feulgen/Sakaguchi ratio decreased with increasing distance from the apex.The following suggestions were made from the results: (a) that there is some degree of quantitative constancy of nuclear arginine which parallels that of DNA; (b) that the amount of nuclear arginine, like that of DNA, is doubled during synthesis in interphase; (c) that the syntheses of DNA and arginine in interphase, if not simultaneous, at least occur within the same relatively short period; (d) that there may be a difference in the DNA/arginine ratio between the nuclei of meristematic and differentiating cells.     

Multiple cysteine proteinase forms during the life cycle of Dictyostelium discoideum revealed by electrophoretic analysis.

Proteinases of the cellular slime mould Dictyostelium discoideum have been analysed using electrophoresis on polyacrylamide gels containing gelatin (gelatin/PAGE). Multiple proteinase forms were apparent in vegetative myxamoebae, but the presence of individual enzyme forms depended on the manner in which the cells were grown. Axenic cells had a characteristic A-pattern of proteinases consisting of six bands, the most active enzymes having apparent Mr values of 51,000 and 45,000 (these have been named ddCP51 and ddCP45, respectively). Some of the proteinases were also present in the medium, the major extracellular form was ddCP42, a 42,000-Mr enzyme. Cells grown in association with bacteria had a distinct B-pattern with three main enzymes that had apparent Mr values of 48,000, 43,000 and 38,000. All of the A- and B-pattern proteinases were most active at acid pH in the presence of dithiothreitol and were inhibited by various agents such as trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E64), leupeptin and chymostatin, which inactivate cysteine proteinases. One of the enzymes, ddCP30, was identified as cysteine proteinase B which had been purified and characterized previously [North, M.J. & Whyte, A. (1984) J. Gen. Microbiol. 130, 123-134]. During starvation of axenic cells in shaken suspensions some of the vegetative proteinases disappeared, ddCP42 was released from the cells and one new enzyme with an apparent Mr of 48,000 appeared. Addition of cyclic AMP had little effect on these changes. When the axenically grown myxamoebae underwent development on filters, similar changes in band pattern were observed and the aggregation stage was characterized by the presence of three cysteine proteinase bands (apparent Mr values of 48,000, 45,000 and 43,000). Proteinases, especially ddCP42, were released from the cells and could be collected from the buffer-saturated pads which supported the filters. The results demonstrate that cysteine proteinases are present throughout growth and development of D. discoideum and that the forms present are subject to nutritional and developmental regulation.     

Adenosine 3′:5′-cyclic monophosphate concentrations and phosphodiesterase activities during axenic growth and differentiation of cells of the cellular slime mould Dictyostelium discoideum

During growth of myxamoebae of Dictyostelium discoideum (strain Ax-2) in axenic medium, the myxamoebae secrete cyclic AMP. As the cells leave the exponential phase of growth and enter the stationary phase, there is an approximate doubling of the intracellular cyclic AMP content, but the amount of extracellular cyclic AMP remains proportional, at all times, to the number of myxamoebae present. During development of axenically grown myxamoebae, there is first a rise in the intracellular concentration of cyclic AMP, followed by a rise in the amount of extracellular cyclic AMP, which reaches a peak at the time of aggregation and then declines. There is a second peak in the amount of extracellular cyclic AMP found at the time of fruiting-body formation, but this second peak is not associated with a rise in the intracellular cyclic AMP concentration. Controls thus exist over the synthesis and secretion of cyclic AMP. Evidence is presented for the belief that the activity of the adenylate cyclase enzyme controls the amount of cyclic AMP synthesized rather than the activity or amount of cyclic AMP phosphodiesterase present. Similar changes occur in extracellular cyclic AMP and phosphodiesterase concentrations during incubation of myxamoebae in buffered suspensions to those occuring during the first few hours of development of such cells on solid media, but the timing of these changes is different.     

Evidence of poly(ADP-ribosylation) in the cockroach Periplaneta americana

Poly(ADP-ribosylation) is a post-translational modification of nuclear proteins typical of most eukaryotic cells. This process participates in DNA replication and repair and is mainly regulated by two enzymes, poly(ADP-ribose) polymerase, which is responsible for the synthesis of polymers of ADP-ribose, and poly(ADP-ribose) glycohydrolase, which performs polymer degradation. The aim of this work was to investigate in the cockroach Periplaneta americana L. (Blattaria: Blattidae) the behaviour of poly(ADP-ribosylation). In particular, we addressed: (i) the possible modulation of poly(ADP-ribosylation) during the embryonic development; (ii) the expression of poly(ADP-ribose) polymerase and glycohydrolase in different tissues; and (iii) the role of poly(ADP-ribosylation) during spermatogenesis. In this work we demonstrated that: (i) as revealed by specific biochemical assays, active poly(ADP-ribose) polymerase and glycohydrolase are present exclusively in P. americana embryos at early stages of development; (ii) an activity carrying out poly(ADP-ribose) synthesis was found in extracts from testes; and (iii) the synthesis of poly(ADP-ribose) occurs preferentially in differentiating spermatids/spermatozoa. Collectively, our results indicate that the poly(ADP-ribosylation) process in P. americana, which is a hemimetabolous insect, displays catalytical and structural features similar to those described in the holometabolous insects and in mammalian cells. Furthermore, this process appears to be modulated during embryonic development and spermatogenesis.     

Subunit structure of the mouse epidermal keratin filament.

The two proteins which are the subunits of mouse epidermal keratin filaments have been isolated from fully differentiated epidermis (stratum corneum), viable differentiating cells and cells grown in culture. The proteins have molecular weights of 68 000 and 60 000, consist of families of very similar species, have common N-terminal (N-acetylserine) and C-terminal (glycine) residues, contain 35--40% alpha-helix and are immunologically cross-reacting. In mixtures, the two proteins polymerize in vitro into native-type keratin filaments that are 70--80 angstrom in diameter, up to 30 micrograms long, possess a characteristic alpha-type X-ray diffraction pattern and contain the subunits in the precise molar ratio of 1 : 2 or 2 : 1.     

Effect of Bacteria on Chemotaxis in the Cellular Slime Molds

The effect of chemotactic substances, secreted by Escherichia coli, on the cellular slime molds was studied by deposition of bacteria near myxamoebae populations. Droplets of a bacterial suspension and a myxamoebae suspension were placed separately, at predetermined distances from each other, on a hydrophobic agar surface of low rigidity. Myxamoebae remained confined inside the droplets, except when they were activated by the bacterial products. The sphere of attraction increased at higher bacterial concentrations. Myxamoebae could be attracted over distances as great as 5 mm. Myxamoebae in droplets close to dense bacterial populations not only were attracted toward the bacteria but also moved out in an opposite direction from the bacteria. There was a gradual decrease of attraction at increasing distances between amoebae and bacteria. The attraction by bacteria or bacterial products was reduced at lower temperatures. Light did not affect the distance over which attraction could be observed. Myxamoebae close to their aggregation phase were most sensitive to the bacterial attractants. Bacterial attractants at high concentrations could disperse aggregates, even when they were in an advanced stage. At still higher concentrations of the bacterial products, cells stopped moving altogether. The bacterial attractants activated different species of cellular slime molds. They appeared to be present not only in E. coli but also in all other bacterial species that were tested. These results are discussed in the light of earlier observations on the attraction of cells by aggregates of myxamoebae.     

Drosophila klaroid encodes a SUN domain protein required for Klarsicht localization to the nuclear envelope and nuclear migration in the eye

KASH (Klarsicht/Anc-1/Syne homology) domain proteins are cytoskeleton-associated proteins localized uniquely to the outer nuclear membrane. Klarsicht is a KASH protein required for nuclear migration in differentiating cells of the Drosophila eye. The C-terminal KASH domain of Klarsicht resides in the perinuclear space, and the cytoplasmic moiety connects to the microtubule organizing center. In C. elegans and vertebrate cells, SUN (Sad1/UNC-84) domain proteins reside in the inner nuclear membrane and tether KASH proteins to the outer nuclear membrane. Is there a Drosophila SUN protein that performs a similar function, and if so, is it like Klarsicht, obviously essential for nuclear positioning only in the eye? Here, we identify Drosophila Klaroid, a SUN protein that tethers Klarsicht. klaroid loss-of-function mutants are indistinguishable phenotypically from klarsicht mutants. Remarkably, neither gene is essential for Drosophila viability or fertility, and even in klaroid klorsicht double mutants, the only obvious external morphological defect is rough eyes. In addition, we find that klaroid and klarsicht are required for nuclear migration in differentiating neurons and in non-neural cells. Finally, while perinuclear Klaroid is ubiquitous in the eye, Klarsicht expression is limited to differentiating cells and may be part of the trigger for apical nuclear migration.     

Amino-acid sequence data of beta-tubulin from Physarum polycephalum myxamoebae

Starting with 7.7 mg of a beta-tubulin isolated from myxamoebae of the slime mould Physarum polycephalum, 90% of the sequence has been determined by the Edman degradation of peptides generated by cyanogen bromide, trypsin and Staphylococcus aureus protease. Differences to other beta-tubulins are mainly conservative and spread evenly throughout the chain except for a high concentration at the C-terminus. The Physarum beta-tubulin shows most homology to Chlamydomonas beta-tubulin (90.5%) and least homology to yeast beta-tubulin (S. cerevisiae, 73.4%). Two tryptic peptides were isolated in approximately equal quantities which were identical except in one position (S/ALTVPELTQRMFDA) showing that at least two beta-tubulins are present in myxamoebae. However, since this was the only heterogeneity found, these beta-tubulins are probably very similar.     

DIFFERENTIATION OF NEUROBLASTOMA, GLIOMA, AND HYBRID CELLS IN CULTURE AS MEASURED BY THE SYNTHESIS OF SPECIFIC PROTEIN SPECIES: EVIDENCE FOR NEUROBLAST-GLIOBLAST RECIPROCAL GENETIC REGULATION

Abstract— Protein species from differentiating neuroblastoma, glioma, and hybrid neuroblastoma-glioma cell lines in cell culture were separated and identified initially in the first dimension by the use of isoelectric focusing gels and were further separated in the second dimension by SDS-acrylamide gels. There were two main classes of proteins identified: proteins which were dominantly expressed in neuroblastoma and also in hybrid cell cultures, and proteins which were expressed in glioma and also hybrid cell cultures. In general, proteins were identified which were significantly expressed in neuroblastoma cells and much reduced in glioma cultures, and also conversely so. The hybrid cell line expressed many of the neuroblastoma-type proteins and relatively fewer of the glioma type proteins. A specific protein species (2) was identified in hybrid cells and was not present in either parental neuroblastoma or glioma cultures. Protein z was expressed however by the co-culturing of neuroblastoma and glioma cells suggesting its induction is dependent on a soluble factor. Protein z in hybrid cells was demonstrated in both stained gels and by autoradiography. Chromosome analysis of hybrid cells confirmed the presence of both rat and mouse chromosomes. It is suggested that similar neuronal-glial interaction may be functional in the intact brain, and that similar reciprocal modulation between neurons and glia may be a central mechanism of differentiation in the nervous system.