Composition,secretion kinetics and radioactive labelling of the extracellular slime polysaccharide secreted during spherulation ofPhysarum polycephalum

The myxomycetePhysarum polycephalum synthesizes copious amounts of slime when differentiating into the hard walled resting stage. The chemical composition of slime obtained after introduction of spherulation in a nutrient and a non-nutrient salt-medium has been analysed and compared. The composition of slime is almost identical after the two different induction methods. This slime could be labelled with radioactived-[U-¹⁴C]glucose,³²PO₄ ^3–,³⁵SO₄ ^2– and⁷⁵SeO₄ ^2–. The kinetics of slime secretion after both induction methods has been followed using different criteria. The sulfate analog⁷⁵SeO₄ ^2– seems to be incorporated into the slime, partially replacing sulfate groups of the sulfated polysaccharide. Furthermore,d-[U-¹⁴C]glucose was used to labe the spherule walls. Determination of an alkali resistent polysaccharide component serves as a new method to follow wall formation.     

Fragmentation of the plasmodium into equally sized pieces by low temperatures in the true slime moldPhysarum polycephalum: A new morphogenesis

Protoplasma - We found a new type of morphogenesis in the plasmodia of the true slime moldPhysarum polycephalum: The plasmodium broke temporarily into pieces with uniform size at low temperatures....     

The life cycle of mitochondria in the true slime mould,Physarum polycephalum

The cytoplasmic aspects of mitochondrial biogenesis have been the focus of much recent attention and a review is presented here of studies on the life cycle of mitochom dria inPhysarum polycephalum. Such studies have focused predominantly on behavior of the mitochondrial genome throughout the mitochondrial life cycle and have been designed to reveal details about (1) the role of the DNA-membrane complex in the segregation of the mitoehondrial genome; (2) the regulation of mitochondrial activity associated with changes in ptoidy of the mitoehondrial nucleus; (3) the hierarchical pattern of transmission of the mitochondrial genome as it relates to the mating-type locus (matA) during the sexual development; and (4) the fusion of mitoehondria that is promoted by a mitochondrial plasmid. The results of such studies contribute significantly to efforts towards a better understanding not only of the mitochondrial life cycle inP. potycephalum but also of the biogenesis of mitoehondria and plastids in many other organisms. Recipient of the Botanical Society Award for Young Scientists, 1988.     

Studies on microplasmodia ofPhysarum polycephalum: II. Fine structure and function of the mucous layer

Summary Microplasmodia of the acellular slime moldPhysarum polycephalum have developed an extensive extracellular slime layer which amounts to 75% of the volume of the total biomass. The slime layer covers the entire plasma membrane including the cell surface invagination system and represents a humid compartment protecting the organism against damaging influence from the surrounding environment.Histochemical treatment with cationic substances revealed that the slime is mostly composed of acid mucopolysaccharides: intensive staining was obtained by colloidal or dialyzed iron, Ruthenium Red, acriflavine and lanthanum hydroxide.At the macromolecular level the slime layer is composed of thin filaments showing a parallel course to the plasma membrane. The slime filaments are very sensitive to fixation and dehydration procedures and tend to form thicker aggregates.From feeding experiments using different fine-particulated markers (Thorotrast, Aerosil) it can be concluded that the slime layer exhibits an important function with regard to the controlled transport and uptake of substances. In particular, the indentations of the cell membrane invagination system running perpendicular from the microplasmodial surface to the cell interior represent preferred pathways for the selective diffusion of macromolecules.     

Sporulation delay by stable cAMP analogues in the slime moldPhysarum polycephalum

Summary Plasmodial cells of the slime moldPhysarum polycephalum become competent for sporulation following a prolonged period of starvation in darkness. Then sporulation can be induced by illumination. Microinjections of the stable (Sp)- and (Rp)-diastereoisomers of adenosine cyclic 3′,5′ monothionophosphate before and during a sensitive period from the start of illumination until 5 h after lead to a significant delay in the sporulation process. Both of the diastereoisomers of cyclic AMP prolong the time for sporangia to form in darkness. However, the (Sp)-diastereoisomer is more effective and causes morphological changes in plasmodia. The experimental data suggest that cyclic AMP is decisively involved in light-induced differentiation in the lower eukaryotoPhysarum polycephalum.     

Two steady states in membrane potential deflection in relation to chemoreception and chemotaxis byPhysarum polycephalum

Summary In the plasmodium ofPhysarum polycephalum application of various monovalent cation salts elicited either a depolarization or a hyperpolarization of the membrane potential. The hyperpolarization was restricted to phosphates and bicarbonates of large cations (Na⁺, Li⁺, NMe₄ ⁺, NEt₄ ⁺). More than 50 other combinations of cations (K⁺, Rb⁺, Cs⁺, NH₄ ⁺) and anions (Cl^–, NO₃ ^–, SO₄ ^2–, acetate, lactate, citrate, etc.) induced the depolarization. In both cases the magnitude of the deflection in membrane potential () varied linearly with logarithm of concentration above the threshold C_th (10^–4 M for all monovalent cation salts examined) according to the following equation: =± R log (C/C_th).The value of R was 10–15 mV, and plus and minus signs correspond to depolarization and hyperpolarization, respectively. Depolarizing and hyperpolarizing agents competed with each other and exhibited a sharp transition between the two states of the membrane which were characterized by — R and R in the above equation or displayed a strong hysteresis, depending on which agents had first been applied to the plasmodial membrane. This transition in the membrane potential corresponded to the transition between positive and negative taxis at the behavioral level.     

Cell membrane isolation from plasmodium ofPhysarum polycephalum

Plasmodia were fractionated to isolate a cell membrane rich fraction by sucrose density-gradient centrifugation. The fractions were identified by electron microscopic observation, PTA-chromic acid staining and assays of marker enzymes, applying the methods for cell membranes of higher plants. The cell membranes were recovered on the density of 1.13 g·cm⁻³.     

Regulation of proteolytic enzymes in axenically grown myxamoebae of Physarum polycephalum

The cultivation of Physarum polycephalum amoebae in two media with different protein contents revealed a regulation of aminopeptidases and proteases depending on the albumin content of the medium: in growing amoebae and plasmodia the aminopeptidases have similar isoenzyme patterns and relative activities against nitroanilides. One alanine and four leucine aminopeptidase isoenzymes were found within the slightly acid pH range. During growth amoebae secrete—different from plasmodia—leucine aminopeptidase into the medium with low protein content. In an albumin-rich medium additional alanine aminopeptidase activity was found. Out of nine plasmodial proteases four were found in amoebae too. Only one band (pI 3.6) was present in the protein-poor medium. No protease activity could be detected in the proteinrich medium.Abbreviations BSA bovine serum albumin - SD semi-defined (medium with low protein content; Table 1) The investigation formed a part of the Ph.D. thesis of A. Haars, Göttingen, 1976     

DNA synthesis in isolated mitochondrial nucleoids from plasmodia ofPhysarum polycephalum

Summary A mitochondrion contains multiple copies of mitochondrial DNA (mtDNA) in the mitochondrial nucleoid (mt-nucleoid, synonym for mitochondrial nuclei). Replicaton of mtDNA in the mtnucleoids appears to be regulated within groups of adjacent mtDNA molecules, known as mitochondrial replicon clusters (MRCs). In this study, we isolated structurally intact mt-nucleoids from the plasmodia ofPhysarum polycephalum and characterized DNA synthesis in the isolated mt-nucleoids. The mt-nucleoids were isolated by dissolving the membranes of highly purified mitochondria with 0.5% Nonidet P-40. The structural integrity of the isolated mt-nucleoid was determined by observing the rod shape of the mt-nucleoid and the structure of the MRC. The isolated mt-nucleoids required four deoxyribonucleoside triphosphates and MgCl₂ for DNA synthesis. The DNA synthesis was resistant to aphidicolin and showed only low sensitivity to N-ethylmaleimide and to ddTTP, suggesting that the DNA synthesis is catalyzed by plant-type mitochondrial DNA polymerase. The capacity for DNA synthesis in the isolated mt-nucleoids was similar to that in the isolated mitochondria, despite removal of most of the mitochondrial matrix and membrane. Furthermore, visualization of sites of DNA synthesis in vitro revealed that DNA synthesis in the isolated mt-nucleoids occurred in each MRC. These results suggest that the isolated mt-nucleoids are capable of efficient and systematic DNA synthesis in vitro. Therefore, the use of isolated mt-nucleoids should permit in vitro characterization of the molecular mechanism of mtDNA replication in the MRC.Abbreviations BrdU 5-bromodeoxyuridine - BrdUTP 5-bromo-deoxyuridine triphosphate - DAPI 4,6-diamidino-2-phenylindole - dNTP deoxyribonucleoside triphosphate - ddCTP dideoxycytidine triphosphate - NEM N-ethylmaleimide - MRC mitochondrial replicon cluster; mt mitochondrial - NP-40 Nonidet P-40 - PBS phosphatebuffered saline - PMSF phenylmethanesulfonyl fluoride - rNTP ribonucleoside triphosphate - VIMPCS video-intensified microscope photon-counting system     

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.     

Behavior of phosphatase isoforms during sclerotium formation in Physarum polycephalum

The behavior of phosphatase isoforms under dark-starvation from plasmodium of Physarum polycephalum were investigated to determine their possible roles in sclerotium formation. Two and a half days after dark-starvation, approximately 95% of plasmodia plates formed sclerotia. Specific phosphatase activity increased markedly up to ca. two-fold within the first day of starvation, after which the enzymatic activity decreased rapidly to a level less than the initial level within 2 days of the starvation period. Among the two isoforms of enzyme detected just before sclerotization under dark-starvation conditions, the enzymatic activity of the major isoform (Rm value of 0.6) decreased gradually within 1.5 days of starvation, then linearly to less than 20% of that at the beginning of the observation. Those of other major isoform (Rm value of 0.7) increased up to ca. two-fold within the first day of starvation, then decreased linearly to levels less than that of the first 2 days of the starvation period. Behavior of this isoform strongly suggests that it initiates the formation of sclerotium under dark-starvation conditions.     

Patterns of oscillation during mitosis in plasmodia ofPhysarum polycephalum

Summary The rhythmic contraction pattern in plasmodia ofPhysarum polycephalum was studied to determine whether characteristic changes occur during the synchronized nuclear division. An electrical method that measures the contraction rhythm in situ during several cell cycles was used. Biopsies of the plasmodia were taken at 17 min intervals for precise determination of the cell cycle stages and were correlated with the simultaneously measured contraction rhythm. All measurements were performed in a temperature controlled environment (27 °C) at 100% relative humidity with the plasmodia (less than 24 h old) growing on a semi-defined agar medium. A total of 14 different plasmodia have been examined, and on one occasion the plasmodium was followed through 3 subsequent mitoses. The mitotic stages were identified with aceto-orcein coloring techniques and by fluorescence methods. Except for a few cases where a mitotic asynchrony of 2–3 min was observed, the mitotic events occurred simultaneously in the nuclei within a single plasmodium. Both the occurrence of the first mitosis after inoculation and the intermitotic times were highly variable. Our study indicates that the contraction rhythm in plasmodia ofPhysarum is unperturbed during the synchronized nuclear division. However, in 5 of the 17 examined mitoses an amplitude decay was observed. We discuss possible explanations for the obtained results with emphasis on the applied techniques, interpretation of the oscillation patterns, and possible restrictions in the cell itself.     

Differential synthesis of an alkaline endonuclease in Physarum polycephalum

During the sclerotization of microplasmodia of Physarum polycephalum in non-nutrient salt medium or in salt medium supplemented by glucose, RNA or nucleotides a 6-fold increase in the specific activity of an alkaline endonuclease was found within 6 h after the induction. The increase was based on de novo synthesis of the enzyme and it was strongly correlated to the sharp drop in the level of cellular RNA in the first hours of the process of scerotization. The induction in exhausted growth medium or in salt medium supplemented by protein or mannitol showed a gradual 2-3-fold increase of the endonuclease in 30 h, parallel to the gradual decrease of the RNA. No changes in the specific activity of the endonuclease were found during logarithmic growth or under conditions of starvation without the induction to sclerotization.The alkaline, polyA-specific endonuclease could possibly regulate the turnover of RNA.     

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.     

Periodic events and cell cycle regulation in the plasmodia ofPhysarum polycephalum

The multinucleated plasmodia ofPhysarum polycephalum, a myxomycete, have been extensively used in cell cycle studies. The natural synchrony of mitosis and DNA synthesis, easy culture methods, the ready fusions obtainable between plasmodia, and the amenability to phase specific studies, employing physical and chemical perturbers, are some of the attractive features of this organism. Because of the absence of a Gl phase in the plasmodia, there is a crowding of cell cycle specific marker events at the G2/M boundary, which reflect features of both the G2/M and the Gl/S boundaries of a typical eukaryotic cell. Prominent among these are the synthesis and overall activity of thymidine kinase, the co-triggering of tubulin and histone genes, translation of their mRNA, the organization and duplication of the microtubular organizing centres of the mitotic spindle and the triggering of cdc 2 kinase activity. These above events have not only served as good markers to monitor the progress of the plasmodial cell cycle, but have also been fairly thoroughly analysed by means of specific perturbers such as DNA synthesis inhibitors, antimicrotubular drugs, UV-irradiation, heat-shock etc. Along with fusion studies, these perturbation studies have been helpful in the formulation of various models on regulation of mitosis. These above aspects as well as prospects for future studies employing this organism are discussed This paper is dedicated to the memory of the late Prof. S C K Nair, formerly University Professor of Physics.     

The twoPhysarum polycephalum profiling are not functionally equivalent in yeast cells

Summary Profilin is a ubiquitous actin-monomer-binding protein. The protistPhysarum polycephalum contains two profilins, ProA and ProP, present in amoebae and plasmodia, respectively. We have used mutantSaccharomyces cerevisiae cells in an attempt to observe distinct functions for the two profilins. Profilin-deficient yeast cells (pfy1) have delocalized actin cortical patches, do not contain visible actin cables, have reduced mating efficiency and do not grow at 37 °C or in the presence of caffeine. Deletion of theSRV2 gene (srv2), coding for the adenylyl cyclase-associated protein, also results in an altered actin distribution and an inability to survive on rich medium. We found that the pfy1 and srv2 mutant phenotypes were corrected equally well by the overexpression of Physarum ProA or yeast Pfy1p profilins. The pfy1 cells overexpressing ProP have improved mating efficiency and a normal distribution of actin cortical patches. These cells, however, have barely detectable actin cables, do not grow at 37 °C, and are sensitive to caffeine. Also, the expression of ProP does not correct the growth defect of the srv2 cells. These results suggest that the two Physarum proteins are not functionally equivalent in yeast cells. No difference was detected in the affinity of ProA and ProP for poly-L-proline, while ProA has a slightly greater affinity than ProP for phosphatidylinositol 4,5-biphosphate.Abbreviations FITC tfluorescein isothiocyanate - PIP₂ phosphatidylinositol 4,5-biphosphate - YPD yeast extract peptone dextrose     

Studies on microplasmodia of Physarum polycephalum

Summary Fragments excised from front regions of thinspread Physarum plasmodia were used to examine a possible correlation between the periodical dynamic activity of such specimens and the spatial organization of actin fibrils. Under isotonic conditions, symmetrical contractions and relaxations of the entire fragment alternate with a period of 1–4 min, whereas under isometric conditions local contractions and relaxations occur simultaneously in different regions of the same specimen. Rapid fixation and phalloidin-staining at distinct stages of the contraction-relaxation cycle demonstrates the permanent existence of cytoplasmic actin fibrils under both isometric and isotonic conditions. During the transition from relaxation to contraction the fibrils shorten in length from 25.5 m to 21.0 m and increase in density from 1.2 fibrils/1000 m² to 2.3 fibrils/1000 m². The present results demonstrate that actin fibrils in Physarum plasmodia are not completely decomposed and reformed every contraction-relaxation cycle.Series Studies on microplasmodia of Physarum polycephalum VIII     

Electron microscopy of the slime moldPhysarum polycephalum

Summary Electron micrographs of fixed sections of the plasmodium of the classic protoplasmic material,Physarum polycephalum, are presented. Numerous round nuclei having well-defined membranes and containing one to three dense nucleoli were especially prominent in the plasmodium. In the surrounding cytoplasm, many irregular membrane-limited bodies were evident, some containing tiny rod-like elements and others with inner structures resembling mitochondria. In addition, there can be seen many small dense granules plus various vacuoles and other inclusions.