Induction of mitochondrial migration in the slime mold Physarum polycephalum

Mitochondrial migration in a microplasmodium of Physarum polycephalumwas studied by litgh and electron microscopy. The mitochondriawere dispersed evenly in the microplasmodium of Physarum polycephalumin shaken cultures but when the microplasmodia were left unshakenin a liquid culture for more than 3 hr, the mitochondria migratedtoward the peripheral area and came into contact with an semi-electrontransparent layer beneath the cell membrane. Once the peripherallocalization of mitochondria was established in unshaken culture,subsequent reversal to the shaken cultures induced a reversion.These results suggest that mitochondrial migration is reversiblyindicated by culture condition. (Received June 19, 1978; )     

Studies on mitochondrial structure and function in Physarum polycephalum. V. Behaviour of mitochondrial nucleoids throughout mitochondrial division cycle

The fine structure of mitochondria and mitochondrial nucleoids in exponentially growing Physarum polycephalum was studied at various periods throughout the mitochondrial division cycle by light and electron microscopy. The mitochondrial nucleoid elongates lingitudinally while the mitochondrion increases in size. When the nucleoid reaches a length of approximately 1.5 mum the mitochondrial membrane invaginates at the center of the mitochondrion and separates the mitochondrial contents. However, the nucleoid does not divide even when the mitochondrial sections are connected by a very narrow bridge. Just before division of the mitochondrion, the nucleoid divides by constriction of the limiting membrane of the dividing mitochondrion. After division, one end of the nucleoid appears to be associated with the inner mitochondrial membrane. The nucleoid then again becomes situated in the center of the mitochondrion before repeating these same processes.     

Cell fusion competence and its induction in Physarum polycephalum and Didymium iridis

In heterothallic Myxomycetes, diploid plasmodia arise when haploid amoebae of two different mating types are cultured together. In this mating process, the amoebae fuse in pairs, and the resulting zygotes develop directly into plasmodia. It has been shown previously that plasmodia start to form in this fashion only when the growing amoebae in a mixed culture reach a critical density. We have investigated the cellular basis of this phenomenon by growing amoebae of different mating type separately from one another and then mixing them to test their mating ability. Amoebae from cultures above and below the critical density were, respectively, competent and incompetent to mate. Furthermore, both partners had to be competent in order for mating to occur. No binucleate cells were formed in mixtures of incompetent amoebae, indicating that they failed to fuse with one another. Incompetent amoebae growing at low density on filters with 0.2-μm pores became competent when the filters were placed on dense cultures of amoebae. We suggest that amoebae release a filter-transmissible material that accumulates during growth and induces the cells to become fusion competent.     

Activity of some dehydrogenase enzymes in mitochondria from Physarum polycephalum.

1. Coupled mitochondria were isolated from exponentially growing Physarum polycephalum. 2. Activity of malate dehydrogenase (oxalacetate reduction) was 10.9 mumol/min/mg protein; the apparent Km was 64 microM. 3. The activity of NADP-isocitric dehydrogenase (IDH) was 110 nmol/min/mg with apparent Km of 35 microM. 4. NAD-IDH showed allosteric properties with AMP as a positive modulator. The apparent Km for the unmodulated activity, 2 mM, was decreased to 0.95 mM by 0.13 mM AMP. 5. Succinic dehydrogenase activity was estimated as three times higher than that of alpha-glycerophosphate dehydrogenase. 6. Mitochondria contained significant amounts of phenolic compounds. Protein estimation by the Bradford method is recommended.     

Reversible inhibition of the hydrocortisone induction of glycerol phosphate dehydrogenase by cytochalasin B in rat glial C6 cells

The hydrocortisone (HC) induction of glycerol phosphate dehydrogenase (GPDH; EC 1.1.1.8) in rat glial C6 cells was inhibited reversibly and in a dose-dependent manner by cytochalasin B (CB). CB had no effect on basal level GPDH, total cellular RNA, DNA or protein content nor did it act as a general inhibitor of the rate of protein synthesis. CB did not appear to be acting via dissociation of microtubules since colcemid had no effect on the induction process. The addition of an alternate energy source (sodium pyruvate) did not relieve the CB inhibition of GPDH induction suggesting that CB is not exerting its effect by blocking glucose utilization. The inhibition by CB is not dependent on the temporal sequence of the induction process since it specifically inhibited GPDH induction at any time it was added. CB did not alter the rate of degradation of GPDH in these cells and direct measurements of the specific rate of synthesis of GPDH demonstrated that CB decreased the induced rate of GPDH synthesis by about 60%. The site of inhibition was more precisely defined by experiments which demonstrated a 60% decrease in specific nuclear binding of ³H-HC even though total cellular uptake of ³H-HC was unaffected. This effect on nuclear binding of HC is sufficient to account for the decreased accumulation of GPDH activity in CB-treated cells.     

Effects of cytochalasin A on the morphology of plasmodia and sclerotia of Physarum polycephalum

Cytochalasin A (CA) at 1.6 X 10(-5)M and lower concentrations produced disruptive effects upon plasmodia, sclerotia, and spherule forms of Physarum polycephalum. CA effects upon either micro- or macroplasmodia included: cytoplasmic condensation, plasmodial contraction, and scission at the plasma membrane. The latter manifestation was most dramatically observed by scanning electron microscopy. Electron microscopy of drug-treated palsmodia confirmed the above phase contrast light-microscopic results and revealed, in addition, vacuolar enlargement, decreased membrane invaginations, and the presence of condensed particles within the plasmodium and at the plasma membrane. These results of drug action were not reversed by washing of exposed plasmodia. Germination of the slcerotial and spherule forms of Physarum was CA-delayed by 12 and 96 hours, respectively. Approximately 10% of drug-treated sclerotia were found to have been burst apart. These dramatic CA effects were nullified by preincubation of the drug either with L-cysteine or with beta-mercaptoethanol; however, iodoacetamide (10(-6)M) gave no such morphologic consequences. Cytochalasins B or D at comparable concentrations were without activity. It is concluded therefore that CA effects upon the myoxomycete reflect specific acceptor responses.     

Specific inhibition of Physarum polycephalum DNA-polymerase-alpha-primase by poly(L-malate) and related polyanions.

Poly(L-malate) is an unusual polyanion found in nuclei of plasmodia of Physarum polycephalum. We have investigated, by enzymatic and fluorimetric methods, whether poly(L-malate) and structurally related polyanions can interact with DNA-polymerase-alpha-primase complex and with histones of P. polycephalum. Poly(L-malate) is found to inhibit the activities of the DNA-polymerase-alpha-primase complex and to bind to histones. The mode of inhibition is competitive with regard to DNA in elongation and noncompetitive in the priming of DNA synthesis. Spermidine, spermine, and histones from P. polycephalum and from calf thymus bind to poly(L-malate) and antagonize the inhibition. The polyanions poly(vinyl sulfate), poly(acrylate), poly(L-malate), poly(D,L-malate), poly(L-aspartate), poly(L-glutamate) have been examined for their potency to inhibit the DNA polymerase. The degree of inhibition is found to depend on the distance between neighboring charges, given by the number of atoms (N) interspaced between them. Poly(L-malate) (N = 5) and poly(D,L-malate) (N = 5) are the most efficient inhibitors, followed by poly(L-aspartate) (N = 6), poly(acrylate) (N = 3), poly(L-glutamate) (N = 8), poly(vinyl sulfate) (N = 3). It is proposed that poly(L-malate) interacts with DNA-polymerase-alpha-primase of P. polycephalum. According to its physical and biochemical properties, poly(L-malate) may alternatively function as a molecular chaperone in nucleosome assembly in the S phase and as both an inhibitor and a stock-piling agent of DNA-polymerase-alpha-primase in the G2 phase and M phase of the plasmodial cell cycle.     

An actin-modulating protein from Physarum polycephalum. I. Isolation and purification

High-speed centrifugation supernatants from slime mould plasmodia show considerable activities to inhibit the polymerization of actin as revealed by viscosity measurements. By following increasing inhibitory activities an actin modulating protein (AM-protein) has been isolated and purified which affects the polymer state of actin. AM-protein has a peptide chain weight of 42 000 and is thus indistinguishable from actin by SDS-electrophoresis, but can be clearly distinguished by isoelectric focussing. Peptide maps from partial proteolytic digests of AM-protein and Physarum actin reveal no similarities thereby excluding that AM-protein is a denatured or modified form of actin. The protein is isolated from crude extracts as a heterodimer with actin to which it strongly binds. This heterodimer affects the polymerization of large amounts of actin by inducing oligomeric or low-polymer actin complexes and thus inhibiting the formation of long actin filaments. The AM-protein/actin heterodimer has only a slight effect of F-actin. It partially depolymerized F-actin within several hours. By ion exchange chromatography in 8 M urea the AM-protein is separated from the actin. The purified AM-protein monomer is renatured and inhibits the polymerization of actin like the heterodimer but additionally, depolymerizes actin filaments very rapidly and effectively by breaking them into oligomer or low-polymer complexes. The addition of less than 1% AM-protein causes a decrease of the specific viscosity of an F-actin solution by 50%. The degree of polymerization inhibition and depolymerization of actin is strictly dependent on the amount of AM-protein added; therefore a catalytic type of reaction between both proteins can be excluded.     

Rosette formation by insect macrophages inhibition by cytochalasin B.

Macrophages from the insect Spodoptera eridania possess membrane receptors for unmodified avian and mammalian erythrocytes, with which they form spontaneous rosettes. Rosette formation occurs in the absence of serum proteins and divalent cations. Individual macrophages bear receptors for several types of red cells. The level of naturally-occurring hemagglutinins against a particular test erythrocyte is not correlated with macrophage reactivity against that red cell. In contrast with mammalian macrophages, neuraminidase treatment of either hemocytes or erythrocytes does not cause a marked enhancement of binding. Pretreatment of macrophages or erythrocytes with cytochalasin B causes reversible inhibition of resetting probably by interfering with normal microfilament function, suggesting that optimal binding occurs when membranes are functioning normally on both macrophages and red cells. Colchicine and vinblastine do not influence resetting; therefore, microtubules are probably not involved in erythrocyte binding.     

Purification and properties of cytoplasmic and mitochondrial malate dehydrogenases of Physarum polycephalum

Two isoenzymes of malate dehydrogenase (MDH) were demonstrated in plasmodia of Physarum polycephalum by polyacrylamide-gel electrophoresis. The more "cathodal" form was uniquely associated with mitochondria (M-MDH) and the other form was found in the soluble cytoplasm (S-MDH). The isoenzymes were separated by acetone fractionation of soluble plasmodial homogenates acidified to pH 5.0. The M-MDH was purified 201-fold by cetylpyridinium chloride treatment, fractionation with ammonium sulfate, gradient elution from sulfoethyl cellulose at pH 6.0, and Sephadex G-100 chromatography. The S-MDH was purified 155-fold by ammonium sulfate fractionation, diethylaminoethyl cellulose chromatography, gradient elution from sulfoethyl cellulose at pH 5.5, and Sephadex G-100 chromatography. The optimal cis-oxalacetate concentrations were 0.35 mM for M-MDH and 0.25 mM for S-MDH, and the optimal pH for both isoenzymes was 7.6 for oxalacetate reduction. The optimal l-malate concentrations were 5 mM for S-MDH and 6 mM for M-MDH, and both isoenzymes exhibited an optimal pH of 10.0 for L-malate oxidation. The Michaelis constants of S-MDH and M-MDH served to discriminate between the isoenzymes. The S-MDH was more heat-stable than the M-MDH. High concentrations of oxalacetate and malate inhibited S-MDH more than M-MDH. The isoenzymes were further distinguished by their utilization of analogues of nicotinamide adenine dinucleotide. Many properties of the Physarum isoenzymes were similar to those of more complex organisms, especially vertebrates.     

Studies on the mechanism of DNA replication in Physarum polycephalum

The synthesis of single-stranded DNA subunits (4 × 10⁷ daltons) in Physarum polycephalum was studied by alkaline sucrose density gradient centrifugation. The results were compared with the synthesis of the double-stranded DNA molecules (2.3 × 10⁸ daltons) which they comprise, as determined from neutral sucrose density gradient centrifugation patterns. Although the initiation of synthesis of most double-stranded DNA molecules takes place relatively early in the S period, synthesis of the subunits within them is initiated throughout at least the first two hours of this period. Similarly, replicating (presumably forked) DNA molecules appear to split into daughter DNA molecules prior to the completion of synthesis of the subunits therein. The average rate of DNA chain elongation within subunits is 0.3 × 10⁶ daltons/minute. It is suggested that alkaline sucrose density gradient centrifugation may be a more sensitive method for determining the time required for the completion of replication than other methods based solely on the incorporation of radioactive DNA precursors into an acid-insoluble product.     

Reversible inhibition of mitochondrial respiratory control by tetrabutylammonium bromide.

Influences of tetrabutylammonium bromide as a phosphorylation inhibitor and as an uncoupler of oxidative phosphorylation in rat liver mitochondria were reversed by washing the organelles. Uncoupling by 2,4-dinitrophenol was also reversible whereas gramicidin and the detergents, sodium dodecylsulfate and cetyltrimethylammonium bromide, were tightly bound uncouplers and they were not substantially removed by simple washing.     

Maternal inheritance of mitochondria: multipolarity, multiallelism and hierarchical transmission of mitochondrial DNA in the true slime mold Physarum polycephalum

Direct evidence of digestion of paternal mitochondrial DNA (mtDNA) has been found in the true slime mold Physarum polycephalum. This is the first report on the selective digestion of mtDNA inside the zygote, and is striking evidence for the mechanism of maternal inheritance of mitochondria. Moreover, two mitochondrial nuclease activities were detected in this organism as candidates for the nucleases responsible for selective digestion of mtDNA. In the true slime mold, there is an additional feature of the uniparental inheritance of mitochondria. Although mitochondria are believed to be inherited from the maternal lineage in nearly all eukaryotes, the mating types of the true slime mold P. polycephalum is not restricted to two: there are three mating loci—matA, matB, and matC—and these loci have 16, 15, and 3 alleles, respectively. Interestingly, the transmission patterns of mtDNA are determined by the matA locus, in a hierarchical fashion (matA hierarchy) as follows: matA7 > matA2 > matA11 > matA12 > matA15/matA16 > matA1 > matA6. The strain possessing the higher status of matA would be the mtDNA donor in crosses. Furthermore, we have found that some crosses showed biparental inheritance of mitochondria. This review describes the phenomenon of hierarchical transmission of mtDNA in true slime molds, and discusses the presumed molecular mechanism of maternal and biparental inheritance.