Amoebae Of Physarum Polycephalum Research Articles

Microtubule-dependent migration of the cell nucleus toward a future leading edge in amoebae ofPhysarum polycephalum

In several cell types, an intriguing correlation exists between the position of the centrosome and the direction of cell locomotion. The centrosome is positioned between the leading edge pseudopod and the nucleus. This suggests that the polarized distribution of organelles in the cytoplasm is coupled spatially with structural and functional polarity in the cell cortex. To study cellular polarization with special interest in the roles of microtubules, we have analyzed the effects of microtubule-disrupting reagents and local laser irradiation on behaviors of both the nucleus and the centrosome in living amoebae ofPhysarum polycephalum. Physarum cells often have 2–3 pseudopods. One of the pseudopods keeps extending to become a stable leading edge while the rest retracts, a crucial step that reorients cells during locomotion. The nucleus, together with the centrosome, moves specifically toward the pseudopod that will become the leading edge. Disruption of microtubules with nocodazole randomizes positions of the nucleus, indicating the involvement of microtubules in the directional migration of the nucleus toward a specific pseudopod. The migration direction of the nucleus is reversed immediately after the UV laser is irradiated at regions between the nucleus and the future leading pseudopod. In contrast, irradiation at regions between the future tail and the nucleus does not affect nuclear migration. By immunofluorescence, we confirmed fragmentation of microtubules specifically in the irradiated region. These results suggest that the nucleus is pulled together with the centrosome toward the future leading-edge pseudopod in a microtubule-dependent manner. Microtubules seem to exert the pulling force generated in the cell cortex on the centrosome. They may serve as a mediator of shape changes initiated in the cell cortex to the organelle geometry in the endoplasm.

Microtubules are required in amoeba chemotaxis for preferential stabilization of appropriate pseudopods.

Amoebae of Physarum polycephalum exhibit chemotactic responses to glucose and to cAMP. The chemotaxing amoebae exhibit alternating locomotive movements: relatively linear locomotion and movements that change the direction of the locomotion. Such locomotive activity is tightly coupled with the changes in the number and the positions of the pseudopods; cells have one pseudopod at the leading edge during their linear locomotion, while they have multiple pseudopods when they are changing the direction of locomotion. Treatment of cells with microtubule-disrupting reagents inhibited the chemotaxis of the cells. To characterize the role of the microtubule system in chemotaxis, we quantitatively analyzed the relationship between the positions of multiple pseudopods of the amoebae and the relative stability of the pseudopods during reorientation. No significant differences were observed in the pseudopod dynamics between the untreated and the treated amoebae. In both cases, one pseudopod at the leading edge continued to expand during linear locomotion. It then split into two to three pseudopods in the reorientation phase, and the positions of the multiple pseudopods were random. Among multiple pseudopods, however, the pseudopods closer to the microneedle tip were selectively stabilized more often than those distant from the tip in the presence of the microtubule system. By contrast, such preferential stabilization of the appropriate pseudopods was completely abolished by microtubule inhibitors. The microtubule-dependent selection of appropriately located pseudopods enables amoebae to turn correctly at the reorientation step.

Two developmentally regulated mRNAs encoding actin-binding proteins in Physarum polycephalum

A cDNA library from amoebae of Physarum polycephalum was screened by differential hybridization. Two clones contained inserts for mRNAs present in amoebae and absent in plasmodia. The LAV3–4 cDNA encodes a 402 aa protein (ABP-46) that shows sequence similarity to the actin binding site in the N-terminal region of the α-actinin family. The LAV3–5 cDNA is 76% identical to the Dictyostelium actin bundling protein, which cross-links and stabilizes actin filaments in amoebal filopodia.

Growth, Development and Genetic Characteristics of Physarum polycephalum Amoebae Able to Grow in Liquid, Axenic Medium

Amoebae from natural isolates of Physarum polycephalum, unlike the plasmodial phase, are unable to grow in axenic medium. A mutant strain of amoebae, CLd-AXE, can be cultured in the liquid, semi-defined medium used for plasmodial culture but lacks some of the properties required for studies of development and gene expression. From crosses of CLd-AXE with wild-type amoebae, new amoebal strains able to grow in axenic medium have been isolated; some of these can also undergo the reversible amoeba-flagellate transformation and apogamic plasmodium development in axenic conditions. Amoebae maintained in active growth in liquid culture for several months showed little change in their properties. Subcultures made with diluted inocula indicated that the same growth rate was achieved even when single amoebae were inoculated in liquid medium. All strains produced colonies with high efficiency when replated on bacterial lawns. Measurements of DNA content by flow cytometry indicated that the majority of amoebae in liquid cultures were haploid. Homozygous diploid amoebae constructed from one strain grew less well than the haploid cells. Genetic analysis of crosses suggested that amoebae able to grow in liquid axenic medium fell into one major phenotypic class with respect to growth rate, and that mutation at only one or two loci was necessary to allow amoebae to grow in axenic medium. Diploid, heterozygous amoebae constructed by mating a mutant with a wild-type strain were unable to grow in axenic medium, indicating that at least one of the putative axe alleles was recessive.

Regulation of Development by the matA Complex Locus in Physarum polycephalum

The transformation of uninucleate amoebae of Physarum polycephalum into multinucleate plasmodia is under the control of a multiallelic mating-type locus, matA. Plasmodium formation usually occurs only when amoebal fusion brings together two different matA alleles within a single cell; such plasmodium formation is termed crossing. Mutations (gadA) in the matA locus permit haploid amoebae to self, that is to form plasmodia in clonal cultures without amoebal fusion. By constructing diploid amoebae heterozygous for gadA alleles, it was shown that the mutant alleles gadA5 and gadA111 were each dominant to gadA +. Non-selfing revertants of gadA mutants may be generated as a result of further mutations (npf) within the matA locus. Sixty-one revertants of this type were studied, derived from three matA3 gadA mutants. Some revertants were able to cross with a matA3 gadA + tester strain. These ‘class I’ revertants failed to cross with one another or with similar revertants of two matA2 gadA mutants; they appear all to lack a single gene function (npfC +) that is necessary in plasmodium development. Other revertants failed to cross with the matA3 gadA + tester. These ‘class II’ revertants also failed to cross with one another, but showed several different patterns of crossing when mixed with other strains. The behaviour of some of the class II revertants suggests that they lack a further gene function (npfB +) necessary in plasmodium development. The gadA mutations may affect a cis-acting regulator of npfB +.

Isolation and characterization of myosin from amoebae of Physarum polycephalum.

Myosin was isolated from amoebae of Physarum polycephalum and compared with myosin from plasmodia, another motile stage in the Physarum life cycle. Amoebal myosin contained heavy chains (Mr approximately 220,000), phosphorylatable light chains (Mr 18,000), and Ca2+-binding light chains (Mr 14,000) and possessed a two-headed long-tailed shape in electron micrographs after rotary shadow casting. In the presence of high salt concentrations, myosin ATPase activity increased in the following order: Mg-ATPase activity less than K-EDTA-ATPase activity less than Ca-ATPase activity. In the presence of low salt concentrations, Mg-ATPase activity was activated approximately 9-fold by skeletal muscle actin. This actin-activated ATPase activity was inhibited by micromolar levels of Ca2+. Amoebal myosin was indistinguishable from plasmodial myosin in ATPase activities and molecular shape. However, the heavy chain and phosphorylatable light chains of amoebal myosin could be distinguished from those of plasmodial myosin in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, peptide mapping, and immunological studies, suggesting that these are different gene products. Ca2+-binding light chains of amoebal and plasmodial myosins were found to be identical using similar criteria, supporting our hypothesis that the Ca2+-binding light chain plays a key role in the inhibition of actin-activated ATPase activity in Physarum myosins by micromolar levels of Ca2+.

Pulsed magnetic fields alter the cell surface

Pulsed magnetic fields (PMFS) are routinely used in the medical community to facilitate bone repair in clinical cases of non-union or pseudarthoses [(1984) Orth. Clin. No. Am. 15, 61-87]. Although this therapeutic regimen appears to be reasonably effective, the mechanism of action between specific PMFs and the target tissue remains unknown. Adding urgency to the need to understand the mechanism are a wide number of reports that have appeared which demonstrate that PMFs similar to those in clinical use can alter many basic physiological functions. We report that a 24 h exposure to PMFs alters the cell surface of Physarum polycephalum amoebae. Further, using the technique of aqueous two-phase partitioning, we present evidence for individual magnetic and electric field, cell surface effects.

Mannosyl glycoprotein(s) function in the conjugation of haploid amoebae of Physarum polycephalum.

Concanavalin A at a concentration of 1.0 mg/ml completely inhibited the conjugation of haploid cells of Physarum polycephalum. This inhibitory effect was counteracted by α-methyl-D-mannoside. α-Mannosidase also inhibited conjugation. Neither wheat germ agglutinin nor lentil lectin had any apparent inhibitory effects. When Physarum cells were cultured in the presence of tunicamycin at a concentration of 1 μ/ml, a similar inhibitory effect on conjugation was observed. The plasma membrane from the amoebae partially inhibited conjugation. On the basis of these results, the mannosyl glycoprotein(s) located on the cell surface is considered to function in conju-gation process.Inhibitory compounds could be extracted from the cell surface with the detergent, Triton CF-54, and could be separated by concanavalin A-Sepharose column chromatography.

Growth and differentiation of wild type amoebae of Physarum polycephalum in liquid culture

A method for growing wild type amoebal strains of Physarum polycephalum in two-membered liquid culture is presented. The medium is a simple buffered salts solution. We found that a minimal level of divalent cation was required for growth. All amoebal strains tried to date have grown under our conditions in stationary culture. Growth under gyratory conditions was only successful at 60 rpm or less and consistent growth required a period of adaptation over several transfers. Differentiation of two apogamic strains, CL and CH1, were compared. Contrary to the results seen on agar plates, the time of onset for the first committed amoebae was identical for both strains in liquid culture. Attempts to demonstrate mating between two genetically compatible amoebal strains grown together in liquid culture were not successful.

A novel acid protease from haploid amoebae of Physarum polycephalum, and its changes during mating and subsequent differentiation into diploid plasmodia.

Haploid myxamoebae of the true slime mold, Physarum poly-cephalum contained an acid protease that had a highly acidic pH optimum of 1.4. This enzyme was not considered to have-SH groups, DAN-Cu2+ reactive-carboxyl groups, or an aspartic acid moiety in its active center. Its activity appeared to decrease with time during mating and the subsequent differ-entiation into the diploid, multinuclear plasmodia.

Purification and characterization of plasma membranes from Physarum polycephalum amoebae

In this study we describe a method for preparing plasma membranes from Physarum polycephalum amoebae. The cells were swollen in hypotonic medium (1 mM ZnCl 2, 10 mM Tris-HCl, pH 8.0) and then broken in a Thomas tissue grinder fitted with a teflon pestle. The plasma membranes were collected by differential centrifugation and purified by centrifugation on a continuous 20–50% sucrose gradient. The membranes sedimented in a single band having a density of 1.16 g/cm 3. They were found by enzymatic assay and by electron microscopy to be free of lysosomes, mitochondria, and nuclei and minimally contaminated by endoplasmic reticulum. The membrane proteins were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis; 10 major and 20 minor bands were seen. Periodic acid Schiff's staining of electrophoretically separated proteins revealed five major and six minor bands containing glycoproteins. Radioiodinated cell surface proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis into six bands with apparent weights greater than 68 000.

Changes in protein and RNA during asexual differentiation of Physarum polycephalum.

Some of the events during the growth and asexual differentiation of Physarum polycephalum amoebae are described. Encysted amoebae contain low levels of protein and RNA. When these cells are mixed with bacteria and inoculated onto agar plates, there is an increase in cellular RNA content followed by an increase in protein content. The cellular RNA and protein contents of all strains tested decrease during the subsequent cell divisions. In nondifferentiating cells (strain Cl at 30 degrees C and strain LU648), RNA and protein contents continue to decrease, and the cell eventually encyst. In the asexually differentiating strain Cl grown at 26 degrees C, the cellular RNA and protein contents stop decreasing and begin to increase when the first amoebae become committed to form plasmodia. At early stages of differentiation a new 36,000 molecular weight polypeptide appears. In fully formed plasmodia another polypeptide of 38,000 molecular is observed. These two plasmodial-specific polypeptides are among the most abundant plasmodial proteins.

Stabilization of monoasters by taxol in the amoebae ofPhysarum polycephalum (Myxomycetes)

Although the plasmodial stage of the MyxomycetePhysarum polycephalum was unaffected with 200 μM taxol, the amoebal stage was sensitive to 10 μM taxol. The first effect of taxol resulted in an accumulation of cells blocked as a monopolar centrosphere surrounded by condensed chromosomes. In 79% of cases these monoasters contained two pairs of centrioles. The mitotic block in a monopolar stage in the presence of taxol delayed the occurrence of late mitotic events such as chromosome decondensation and formation of the nuclear envelope. Escape from the monopolar centrosphere stage and formation of multinucleated amoebae involved a transient monopolar reconstruction stage in which a long microtubular bundle interacted with a small chromosomal mass outside the monoaster.

Thymidine-dependent growth of Physarum polycephalum amoebae treated with methotrexate in axenic culture

Low levels of methotrexate block the growth of Physarum amoebae in axenic culture. The block is readily reversed by adding thymidine to the medium.

TWO MULTIALLELIC MATING COMPATIBILITY LOCI SEPARATELY REGULATE ZYGOTE FORMATION AND ZYGOTE DIFFERENTIATION IN THE MYXOMYCETE PHYSARUM POLYCEPHALUM

The mating of Physarum polycephalum amoebae, the ultimate consequence of which is a "plasmodium," was recently shown to be governed by two compatibility loci, matA (or mt) and matB (Dee 1978; Youngmanet al. 1979). We present evidence that matA and matB separately regulate two discrete stages of mating: in the first stage, amoebae (which are normally haploid) fuse in pairs, with a specificity determined by matB genotype, to form diploid zygotes; subsequent differentiation of the zygotes into plasmodia is regulated by matA and is unaffected by matB. Mixtures of amoebae carrying unlike matA and matB alleles formed diploids to the extent of 10 to 15% of the cells present, and the diploids differentiated into plasmodia. When only the matB alleles differed, diploid cells still formed to a comparable (5 to 10%) extent, but rather than differentiating, these diploids remained amoebae. When strains carried the same alleles of matB, formation of diploid cells was greatly reduced: in like-matB, like-matA mixtures, none of 320 cells examined was diploid; in like-matB, unlike mat-A mixtures, differentiating diploids could be detected, but at only 10(-3) to 10(-2) the frequency of unlike-matB, unlike-matA mixtures. The nondifferentiating diploid amoebae recovered from unlike-matB, like-matA mixtures were genetically stable through extensive growth, even though they grew more slowly than haploids (10-hr vs. 8-hr doubling period), and could be crossed with both haploids and diploids. The results of such higher ploidy and mixed ploidy crosses indicate that karyogamy does not invariably accompany zygote formation and differentiation.

Effect of microtubule-disrupting drugs on protein and RNA synthesis in Physarum polycephalum amoebae.

The effects of the microtubule-disrupting drugs, colchicine, vinblastine, podophyllotoxin, griseofulvin, and lumicolchicine (10(-5) M), on protein and RNA synthesis were studied in Physarum polycephalum amoebae in culture. All, except lumicolchicine, were found to simultaneously reduce the rate of protein synthesis and stimulate RNA synthesis. These results parallel the effects seen in cells exposed to heat shock. Treatment of cells with a microfilament-disrupting drug, cytochalasin B (10 micrograms/ml in ethanol), resulted in a reduced rate of protein synthesis after 2 h compared to a similar effect by vinblastine in 5--15 min. A morphological abnormality, microtubule paracrystals, were seen associated with centrioles in vinblastine-treated cells in which protein synthesis had been reduced by 50%. Vinblastine and podophyllotoxin were shown to interfere with the recovery of protein synthesis after inhibition by low or elevated temperatures. The possible role of microtubules in regulating the translational response of a cell to an external environmental stimulus is discussed.

The structure of the pro-flagellar apparatus of the amoebae ofPhysarum polycephalum: Relationship to the flagellar apparatus

Unflagellated amoebae ofPhysarum polycephalum (Myxomycete) possess a pro-flagellar apparatus. A tridimensional ultrastructural model of the pro-flagellar apparatus is proposed, based upon observations of thin sections of whole amoebae and of isolated nucleo-pro-flagellar apparatus complexes. The pro-flagellar apparatus is composed by the same basic elements as the fully differentiated flagellar apparatus and differs from the latter by three main aspects: the two kinetosomes of the pro-flagellar apparatus are devoid of flagella; the posterior kinetosome is directed forward, and, with the exception of microtubular arrays 1 and 5, all the other microtubular arrays (2–4) have a reduced length, although they show a normal number of constituting microtubules. We demonstrate the existence of an extension of the posterior para-kinetosomal structure which is present both in the pro-flagellar and in the flagellar apparatus. The growth of the microtubules of the pericentriolar arrays and of the flagella could be the main event which leads to the formation of the fully developed flagellar apparatus inPhysarum amoebae.

Locomotion of Physarum polycephalum amoebae is guided by a short range interaction with E. coli

Studies of the behavior of Physarum polycephalum amoebae have shown that locomotion of these cells is guided by surfaces composed of aggregated bacteria. Amoebae move readily on both E. coli and agar surfaces. However, when a cell migrating on bacteria encounters an edge of the bacterial surface, the orientation of cell movement changes so that the cell maintains contact with bacteria. Time-lapse cinemicrographic studies employing wild type and mutant cells show that this behavior involves short range interactions between amoebae and bacteria, that it is not dependent on variations in the rate of phagocytosis, and that it is not a simple result of constraints on cell movement imposed by adhesive bonds between amoebae and bacteria. These results provide evidence that guidance of cell locomotion depends on active regulation of the cellular force generating system as the amoebae contact surfaces of varying characteristics and, therefore, suggest that this system is amenable to detailed studies of process involved both in cell-cell recognition and in linking such recognition to regulation of cell movement.

The role of regulation of cell speed in the behavior of Physarum polycephalum amoebae

Studies on the behavior of wild-type and mutant Physarum polycephalum amoebae have revealed that regulation of cell speed results in different patterns of cell dispersion in different environments and have shown that P. polycephalum can be used for genetic studies of the mechanisms responsible for this element of cell behavior. Colonies generated by clonal populations of amoebae growing on E. coli display alternate colony morphologies depending on the pH of the culture medium and the presence of live E. coli as a nutrient. In the larger ‘spreading colonies’ cells at the outside of a colony are dispersed over a wide band of bacteria while in the smaller ‘aggregate ring colonies’ most cells moving on bacteria are aggregated in a regularly shaped ring on a narrow band of bacteria at the border of the bacterial lawn created when amoebae completely consume the bacteria available in the colony center. Measurements of cell growth, the rate of colony expansion, and the rate of single cell movement show that cells in contact with bacteria move more slowly in aggregate ring than in spreading colonies. Moreover, since in aggregate ring colonies the rate of movement of cells in contact with bacteria is also reduced relative to that of cells moving on adjacent regions of the agar surface, inhibition of cell speed appears to be at least partially responsible for generating the aggregate ring morphology. Characterization of the behavior of a single locus mutant which generates spreading colonies under conditions where aggregate ring colonies are normally formed has provided additional evidence that a specific mechanism is involved in controlling the distribution of amoebae through regulation of cell speed. Furthermore, the studies of this mutant have shown that aberrant colony morphology can be used as an easily recognized phenotype for identifying and studying mutants with defects which affect the regulation of cell speed.

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.