Changes in membrane glycoproteins during fruit-body formation inPhysarum polycephalum

Sporangia formation ofPhysarum polycephalum was induced by starvation and illumination, and the morphogenic process during the differentiation was studied by scanning electron microscopy. Plasma membranes were prepared from these differentiating plasmodia and the membrane proteins were analyzed by polyacrylamide gel electrophoresis. Many glycoproteins appeared during the fruit-body formation. Of these a protein of apparent molecular mass of 66 kD was prominent in sporangia forming stage which showed a high affinity to RCA lectin. Inhibition of the glycosylation and processing of these glycoproteins resulted in the prevention of fruit-body formation suggesting that the synthesis of these membrane components is a prerequisite process for the sporangia formation in the slime mold.     

Spherule wall formation inPhysarum polycephalum

Summary Microplasmodia ofPhysarum polycephalum Schw differentiate into numerous hard walled spherules when deprived of nutrients. Cleavage occurs by invagination of the plasmalemma and growth of furrows centripetally. Cleavage furrows consistently contain ruthenium red positive fibrillar material. As the spherules become more rounded, a compact layer of ruthenium red positive material surrounds their membranes. A spherule wall which does not bind ruthenium red is deposited inside of the stained layer. Clusters of spherules are enclosed by ruthenium red positive material similar to the microplasmodial slime.Partially supported by a grant from Research Corporation.     

Studies on cyst formation inPhysarum polycephalum myxamoebae

Encystment of myxamoebae ofPhysarum polycephalum was induced by transferring the amoebae to a high salt medium of 1/60 M Sørensen buffer (pH 6.0) containing 0.125 M NaCl, 1.6 mM MgCl₂ and 0.18 mM CaCl₂. The induction of cysts was blocked by inhibitors of protein synthesis, such as puromycin, cycloheximide and streptomycin. However, inhibitors of RNA synthesis, such as actinomycin D, proflavin and 8-azaguanine did not block the transformation. These results suggest that in the cyst formation,de novo RNA synthesis is not involved, whereas protein synthesis is required. Cyst formation was more strongly inhibited by inhibitors of oxidative phosphorylation than by other respiratory poisons. It seems that oxidative phosphorylation takes part in the energy supply of this differentiation.     

Change in ATP-pyrophosphohydrolase activity during spherule formation of Physarum polycephalum.

The activity of Ca2+-dependent ATP pyrophosphohydrolase was found to fluctuate during spherule formation of the acellular slime mold Physarum polycephalum under starving incubation. The enzyme activity increased up to 16-fold at the 3rd day of the starvation, then decreased drastically to less than its original level. Column chromatography of the enzyme preparation suggested that the increase in the activity was due to de novo synthesis of a new isozyme. Cycloheximide inhibited the synthesis. The two isozymes were different in their Ca2+ sensitivity, the new one being less sensitive.     

Induction of spherule formation in Physarum polycephalum by polyols

A method has been developed for inducing spherule formation (spherulation) in the myxomycete Physarum polycephalum by transferring the culture to synthetic medium containing 0.5 m mannitol or other polyols. This morphogenetic process occurred within 12 to 35 hr after the inducer was added. The mature spherules existed as distinct morphogenetic units, in contrast to the clusters of spherules formed during starvation. Ninety per cent of the spherules germinated by 24 hr in synthetic medium. The changes in the synthesis of ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and protein during plasmodial growth, spherulation, and germination of spherules are described. When spherule formation was completed, RNA, protein, and DNA decreased, compared with the values at the beginning of the conversion. The incorporation of (3)H-uridine into trichloroacetic acid-insoluble material was different in each of these periods, and this incorporation was sensitive to actinomycin D. The amount of glycogen increased during growth, whereas it decreased during spherulation. (14)C-glucose could be taken up by the cells in the presence of the inducer, and mannitol could not replace glucose as a source of energy. The mode of action of mannitol and its mechanism of induction are discussed.     

Changes in the activity of selected enzymes during starvation of Physarum polycephalum

The levels of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, lactate dehydrogenase, and cyclic phosphodiesterase activities were examined in growing and starving plasmodia of Physarum polycephalum. The activities of lactate dehydrogenase, 6-phosphogluconate dehydrogenase, and glucose-6-phosphate dehydrogenase decreased whereas that of cyclic phosphodiesterase increased. The change in activity of lactate dehydrogenase was the result of the variation of the activity of a single enzyme quite similar to the lactate dehydrogenases of higher animals.     

Phage formation in Staphylococcus muscae cultures; nucleic acid synthesis during virus formation

1. The total nucleic acid synthesized by normal and by infected S. muscae suspensions is approximately the same. This is true for either lag phase cells or log phase cells. 2. The amount of nucleic acid synthesized per cell in normal cultures increases during the lag period and remains fairly constant during log growth. 3. The amount of nucleic acid synthesized per cell by infected cells increases during the whole course of the infection. 4. Infected cells synthesize less RNA and more DNA than normal cells. The ratio of RNA/DNA is larger in lag phase cells than in log phase cells. 5. Normal cells release neither ribonucleic acid nor desoxyribonucleic acid into the medium. 6. Infected cells release both ribonucleic acid and desoxyribonucleic acid into the medium. The time and extent of release depend upon the physiological state of the cells. 7. Infected lag phase cells may or may not show an increased RNA content. They release RNA, but not DNA, into the medium well before observable cellular lysis and before any virus is liberated. At virus liberation, the cell RNA content falls to a value below that initially present, while DNA, which increased during infection falls to approximately the original value. 8. Infected log cells show a continuous loss of cell RNA and a loss of DNA a short time after infection. At the time of virus liberation the cell RNA value is well below that initially present and the cells begin to lyse.     

Behavior of mitochondria and their nuclei during amoebae cell proliferation inPhysarum polycephalum

Summary We investigated the manner of mitochondrial DNA (mtDNA) replication and distribution during the culture ofPhysarum polycephalum amoebae cells by microphotometry, anti-BrdU immunofluorescence microscopy, and quantitative hybridization analysis. In amoebae cells ofP. polycephalum, the number of mitochondria per cell and the shape of both mitochondria and mitochondrial nuclei (mt-nuclei) noticeably changed over the culture period. At the time of transfer, about 27 short ellipsoidal shaped mitochondria, which each contained a small amount of DNA, were observed in each cell. The number of mitochondria per cell decreased gradually, while the amount of mtDNA in an mt-nucleus and the length of mt-nuclei increased gradually. Midway through the middle logarithmic growth phase, the number of mitochondria per cell reached a minimum (about 10 mitochondria per cell), but most mtnuclei assumed an elongated shape and contained a large amount of mtDNA. During the late log- and stationary-growth phase, the number of mitochondria per cell increased gradually, while the amount of DNA in an mt-nucleus and mt-nuclei length decreased gradually. Upon completion of the stationary phase, the number and condition of mitochondria within cells returned to that first observed at the time of transfer. The total amount of mtDNA in a cell increased about 1.6-fold the first day, decreased immediately, then maintained a constant level ranging from 130 to 160 T. Except for the fact that mtDNA synthesis began earlier than synthesis of cell nuclei, the rate of increase in mtDNA paralleled that of cell-nuclear DNA throughout the culture. These results indicate that mtDNA is continuously replicated in pace with cell proliferation and the rate of mitochondrial division varies during culture; this mitochondrial division does not synchronize with either mtDNA replication or cell division. Furthermore, we observed the spatial distribution of DNA replication sites along mt-nuclei. Replication began at several sites scattered along an mt-nucleus, and the number of replication sites increased as the length of mt-nuclei increased. These results indicate that mtDNA replication progresses in adjacent replicons, which are collectively termed a mitochondrial replicon cluster.Abbreviations DAPI 4,6-diamidino-2-phenylindole - VIMPCS video-intensified microscope photon counting system - BrdU 5-bromodeoxyuridine - FITC fluorescein isothiocyanate     

Study of nuclear basic proteins of Physarum polycephalum: Differential synthesis during plasmodial cell cycle and spherule germination

An electrophoresis system providing a good resolution of all basic proteins and their variants or modified forms extracted from Physarum polycephalum nuclei has been used to study the synthesis of the basic nuclear proteins of this organism during three developmental stages: the S-phase and the G2-phase of the plasmodial cell cycle and the premitotic stage of germinating spherules. Differences have been seen in the synthesis of the different classes of histones and HMG like proteins during these three stages.     

Cytoskeletal changes visualized by fluorescence microscopy during amoeba-to-flagellate and flagellate-to-amoeba transformations inPhysarum polycephalum

Summary Changes in the intracellular distribution of microtubules and microfilaments during amoeba-to-flagellate and flagellate-to-amoeba transformations inPhysarum polycephalum were examined by fluorescence microscopy using anti-tubulin antibody and NBD-phallacidin, respectively. Amoebae contained an extensive microtubular cytoskeleton, which was converted to a flagellar cone structure during transformation to flagellates in liquid medium. When flagellates reverted back to amoebae, this conical structure disintegrated prior to flagella resorption. Amoebae showed some microfilament-enriched domains along the periphery, from which numerous filamentous extrusions, probably pseudopods and filopods, emanated. Flagellates contained a ridge, a sheet-like structure, along their dorsal axis, especially in the earlier stages of flagellation. Another microfilament-enriched thick filamentous structure ran along the dorsal axis, starting from the anterior tip of the cell. This structure apparently coincided spatially with one of the bundles of microtubules. During the reversion to amoebae, other localized microfilaments were transiently observed at the posterior end. A model of cytoskeletal changes in the transformations between these two cell types was proposed.     

A gene,imz, affecting the pH sensitivity of zygote formation inPhysarum polycephalum

Mating inPhysarum polycephalum involves the fusion of two haploid amoebae and the differentiation of the resulting diploid zygote into a multinucleate plasmodium. Mating proceeds optimally with amoebae growing on an agar medium at pH 5.0. At pH 6.2, the amoebae still grow normally, but mating is completely blocked. The barrier at pH 6.2 is not in the differentiation step, since preformed diploids readily convert to plasmodia at this pH. The barrier can be overcome by raising the ionic strength of the agar medium; the effect, moreover, is not ion-specific. We have discovered a genetic locus,imz (ionicmodulation of zygote formation), that affects the upper pH limit for mating; the respective limits associated with the two known alleles,imz-1 andimz-2, are pH 5.6 and pH 6.0 at low ionic strength. Animz-1×imz-2 mating displays the pH 6.0 limit;imz-2 is therefore “dominant”. We suggest that this new gene affects a cell component that is exposed to the exterior of the amoeba and is involved in the fusion step of mating.     

Magnesium starvation of Aerobacter aerogenes. I. Changes in nucleic acid composition

Aerobacter aerogenes incubated in a medium containing all factors necessary for exponential growth except Mg⁺⁺ continued to synthesize nucleic acids and proteins for more than 70 hr, provided the major carbon source was in excess at all times. After 24 hr of Mg⁺⁺ starvation, deoxyribonucleic acid content in the culture had increased 10-fold. In contrast, the viable-cell count increased only about threefold during the first few hours and then remained approximately constant for the subsequent 70 hr. After specified intervals of Mg⁺⁺ starvation, extracts of the bacteria, or ribonucleic acid (RNA) purified from them, was centrifuged through gradients of sucrose to separate transfer RNA from ribosomal components. After correcting for losses, we obtained the following results. (i) There was a progressive rise in the content of transfer RNA competent to accept amino acids and during starvation it remained completely stable. (ii) In contrast, the contents of normally sedimenting ribosomal RNA and ribosomal subunits (30 and 50S) remained approximately constant for more than 24 hr. This did not result from stability of ribosomes made prior to starvation together with an inhibition of synthesis of new particles. Rather, ribosomes were continually breaking down and being replaced by an equivalent number of new ones. (iii) The breakdown of ribosomes appeared to be sequentially ordered; polysomes yielded 70S monomers, which then gave 30 and 50S particles, and these disintegrated to smaller units and finally to acid-soluble products. (iv) Furthermore, the particles derived from breakdown do not appear to exchange with subparticles on the path of assembly. Thus, ribosome decay was age-dependent and ribosomal RNA molecules had a minimal life expectancy of 90 min; however, some survived much longer.     

Changes in lipid composition during starvation and germ-tube formation inCandida albicans

The changes in the lipid components ofCandida albicans have been determined during growth, starvation, and germ-tube formation.¹⁴C-Labeled cells were used to determine the extent of synthesis and degradation of the different lipid fractions. On a dry weight basis the percentage of total lipid increased from 18% for blastospores to 22% after starvation but decreased to 11% after 4 h of germination. The major components of the lipid fraction were sterols (36–52%) and phospholipids (28–42%). The free and esterified sterol fractions both increased approximately 45% during starvation. The free sterol content continued to increase over the initial stage of germ-tube formation, then decreased with time. The sterol ester fraction decreased throughout germination to the concentration found in growing blastospores. The changes in specific activity (cpm/μg sterol) of these fractions indicated that sterol esters were precursors for free sterols during germination. The total phospholipid fraction increased during starvation but there was a decrease in both the cellular concentration (60%) and the specific activity during germination. There were only minor changes in the relative concentrations of phosphatidylinositol, phosphatidylserine, phosphatidylcholine, and phosphatidylethanolamine which indicated coordinate synthesis and degradation of these components. Free fatty acids and triacylglycerides are only minor components of the total lipid pool.     

Effect of saffron on cell colony formation and cellular nucleic acid and protein synthesis.

A concentrated extract of saffron was prepared from the flowers of Crocus sativis. The effect of this extract on the ability of HeLa cells to form colonies, and on cellular DNA, RNA and protein synthesis was examined. Incubation of cells with extract for 3 h resulted in significant inhibition of colony formation and cellular nucleic acid synthesis with 50% inhibition at concentrations of approximately 100-150 micrograms/ml. In contrast there was no inhibition of cellular protein synthesis at concentrations of extract as high as 400 micrograms/ml.     

Growth and development in relation to the cell cycle inPhysarum polycephalum

Summary In strain CL ofPhysarum polycephalum, multinucleate, haploid plasmodia form within clones of uninucleate, haploid amoebae. Analysis of plasmodium development, using time-lapse cinematography, shows that binucleate cells arise from uninucleate cells, by mitosis without cytokinesis. Either one or both daughter cells, from an apparently normal amoebal division, can enter an extended cell cycle (28.7 hours compared to the 11.8 hours for vegetative amoebae) that ends in the formation of a binucleate cell. This long cycle is accompanied by extra growth; cells that become binucleate are twice as big as amoebae at the time of mitosis. Nuclear size also increases during the extended cell cycle: flow cytometric analysis indicates that this is not associated with an increase over the haploid DNA content. During the extended cell cycle uninucleate cells lose the ability to transform into flagellated cells and also become irreversibly committed to plasmodium development. It is shown that commitment occurs a maximum of 13.5 hours before binucleate cell formation and that loss of ability to flagellate precedes commitment by 3–5 hours. Plasmodia develop from binucleate cells by cell fusions and synchronous mitoses without cytokinesis.Abbreviations CL Colonia Leicester - DSDM Dilute semi-defined medium - FKB Formalin killed bacterial suspension - IMT Intermitotic time - LIA Liver infusion agar - SBS Standard bacterial suspension - SDM Semi-defined medium