Differences in the surface properties of the mating types of Physarum polycephalum

In the slime mold Physarum polycephalum, formation of a diploid plasmodium occurs when compatible haploid amoebae fuse. To study cell surface changes associated with the fusion process, a non-destructive method known as aqueous, two-phase partitioning was employed. Using a two-phase system of dextran and polyethylene glycol, we observed that the two mating types (RSD4 and MA185) have different partition coefficients and hence different surface properties. Based on their partitioning behavior, MA185 cells appear to have a more hydrophobic surface than RSD4 amoeba. The partition coefficient of both cell types decreased with time. If amoebae were maintained in culture until they encysted, differences in their surface were not detectable.     

Inhibition of growth and cell wall morphogenesis of Bacillus subtilis by extracellular slime produced by Physarum flavicomum.

Slime secreted by microplasmodia of the myxomycete Physarum flavicomum inhibited the uptake of glucose and amino acids, as well as growth and cell division of the bacterium Bacillus subtilis. Morphological changes such as production of chains, swollen cells, and/or cell lysis, occurred coincident with these physiological inhibitory events. These phenomena were all dependent on the concentration of slime present in the growth medium. Electron microscopy revealed that the cell walls of slime-inhibited cells were undergoing degradation and the process was most pronounced in the swollen cells. Isolated cell walls of B. subtilis were also found to undergo degradation upon incubation with slime. Boiled slime did not exhibit lytic activity on native cell walls, but boiled cell walls were degraded by native slime. The inhibitory effect of slime seemed to be, at least in part, due to an inherent peptidase (protease) activity. B. subtilis eventually overcomes the inhibition exhibited by slime due to the production of an antagonist of slime.     

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     

Cellular events during sexual development from amoeba to plasmodium in the slime mould Physarum polycephalum

Time-lapse cinematography and immunofluorescence microscopy were used to study cellular events during amoebal fusions and sexual plasmodium development in Physarum polycephalum. Amoebal fusions occurred frequently in mixtures of strains heteroallelic or homoallelic for the mating-type locus matA, but plasmodia developed only in the matA-heteroallelic cultures. These observations confirmed that matA controls development of fusion cells rather than cell fusion. Analysis of cell pedigrees showed that, in both types of culture, amoebae fused at any stage of the cell cycle except mitosis. In matA-heteroallelic fusion cells, nuclear fusion occurred in interphase about 2 h after cell fusion; interphase nuclear fusion did not occur in matA-homoallelic fusion cells. The diploid zygote, formed by nuclear fusion in matA-heteroallelic fusion cells, entered an extended period of cell growth which ended in the formation of a binucleate plasmodium by mitosis without cytokinesis. In contrast, no extension to the cell cycle was observed in matA-homoallelic fusion cells and mitosis was always accompanied by cytokinesis. In matA-homoallelic cultures, many of the binucleate fusion cells split apart without mitosis, regenerating pairs of uninucleate amoebae; in the remaining fusion cells, the nuclei entered mitosis synchronously and spindle fusion sometimes occurred, giving rise to a variety of products. Immunofluorescence microscopy showed that matA-heteroallelic fusion cells possessed two amoebal microtubule organizing centres, and that most zygotes possessed only one; amoebal microtubule organization was lost gradually over several cell cycles. In matA-homoallelic cultures, all the cells retained amoebal microtubule organization.     

Heterovesicula cowani N. G., N. Sp. (Heterovesiculidae N. Fam.), a Microsporidian Parasite of Mormon Crickets, Anabrus simplex Haldeman, 1852 (Orthoptera: Tettigoniidae)

ABSTRACT. Heterovesicula cowani , n. g., n. sp., is a dimorphic microsporidium described from the adipose tissue of the Mormon cricket, Anabrus simplex Haldeman. Proliferation of the microsporidium is by karyokinesis of uninucleate and binucleate cells to form binucleate and tetranucleate cells, respectively. These cells will undergo binary fission (merogony). Ultimately, the meronts undergo karyokinesis without subsequent cytokinesis producing spherical multinucleate plasmodia that are transitional to 2 types of sporogony. Transitional to disporoblastic sporogony, a fragile interfacial envelope delaminates from the plasmodium with morphogenesis to a monfiliform plasmodium consisting of fusiform binucleate diplokaryotic sporonts. These undergo karyokinesis to form tetranucleate diplokaryotic sporonts that undergo cytokinesis during disintegration of the plasmodium into isolated binucleate sporonts. Transitional to octosporoblastic sporogony, multinucleate plasmodia disintegrate into short monofiliform plasmodia of diplokaryotic sporonts which then segregate while undergoing gradual nuclear dissociation (haplosis by nuclear dissociation). These undergo two sequences of karyokinesis and subsequent multiple fission to form eight uninucleate (haploid) sporoblasts in a fusiform arrangement within a persistent envelope. Binucleate spores are ovocylindrical, about 5.4 × 1.7 μm (fresh), with an isofilar polar filament singly coiled about 11 turns. Uninucleate spores are ovoid to slightly pyriform, 4.0 × 1.7 μm (fresh), with an isofilar filament singly coiled about 9 turns. A new family, Heterovesculidae, is proposed for the new genus.     

Expression of poriferasterol monoglucoside associated with differentiation of Physarum polycephalum

A glycolipid which was expressed during a differentiation from haploid myxoamoebae to diploid plasmodia of a true slime mold, Physarum polycephalum, has been examined. In the amoeboid stage, cells did not contain this glycolipid, but after conjugation of the haploid cells, this substance appeared and increased in its amount. From structural studies of the purified glycolipid, it has been identified as poriferasterol monoglucoside.     

Effects of an acridine half-mustard (ICR 191) on growth and ploidy of frog cells in culture

The effects of an acridine half-mustard, ICR 191, on the growth rate and ploidy of four haploid and two diploid lines of Rana pipiens cells in culture were studied. Growth curves indicate that the haploid and diploid cell lines were equally resistant to a 4-hour exposure of this drug (0.1 micrometer to 10 micrometer. ICR 191 treatment induced the haploid cell cultures to become diploid. The proportion of diploid cells increased progressively with respect to time after the 4-hour exposure period. The greater the concentration of ICR 191 applied, the more rapid the rate of conversion. Autoradiographic determinations of percent labelled nuclei indicate that DNA synthesis was not inhibited in haploid or in diploid cells. Therefore, the increased proportion of diploid cells did not originate from the small percentage of diploid cells in the initial population. Instead the haploid cells were converted to diploid cells. Time lapse cinematography indicated that the conversion mechanism was other than cell fusion. Conversion to higher ploidy did not occur when diploid cell cultures were exposed to ICR 191.     

Inheritance of extrachromosomal rDNA in Physarum polycephalum.

In the acellular slime mold Physarum polycephalum, the several hundred genes coding for rRNA are located on linear extrachromosomal DNA molecules of a discrete size, 60 kilobases. Each molecule contains two genes that are arranged in a palindromic fashion and separated by a central spacer region. We investigated how rDNA is inherited after meiosis. Two Physarum amoebal strains, each with an rDNA recognizable by its restriction endonuclease cleavage pattern, were mated, the resulting diploid plasmodium was induced to sporulate, and haploid progeny clones were isolated from the germinated spores. The type of rDNA in each was analyzed by blotting hybridization, with cloned rDNA sequences used as probes. This analysis showed that rDNA was inherited in an all-or-nothing fashion; that is, progeny clones contained one or the other parental rDNA type, but not both. However, the rDNA did not segregate in a simple Mendelian way; one rDNA type was inherited more frequently than the other. The same rDNA type was also in excess in the diploid plasmodium before meiosis, and the relative proportions of the two rDNAs changed after continued plasmodial growth. The proportion of the two rDNA types in the population of progeny clones reflected the proportion in the parent plasmodium before meoisis. The rDNAs in many of the progeny clones contained specific deletions of some of the inverted repeat sequences at the central palindromic symmetry axis. To explain the pattern of inheritance of Physarum rDNA, we postulate that a single copy of rDNA is inserted into each spore or is selectively replicated after meiosis.     

Microelectrode studies of oxygen transfer in trickling filter slimes*

Slime-covered rocks and samples of process waters from two trickling filters for treatment of municipal wastes were brought to the laboratory for probing with microelectrodes to determine dissolved oxygen (DO). Slime thickness was 0.4–1.5 mm. Flow rate of medium over the slime had a minor effect on slime respiration, but pH 5 or below was strongly inhibitory. Increasing temperature showed lower oxygen concentration throughout a slime, although 27°C had results little different from those at 22°C. Medium concentration had a profound effect on oxygen concentration profiles, and either oxygen-limited or substrate limited respiration could be demonstrated. Illumination of slimes from the top of the trickling filter developed oxygen supersaturation because oxygen from photosynthesis could not diffuse away rapidly.     

Nuclear migration in Saccharomyces cerevisiae is controlled by the highly repetitive 313 kDa NUM1 protein.

We have isolated a novel gene (NUM1) with unusual internal periodicity. The NUM1 gene encodes a 313 kDa protein with a potential Ca2+ binding site and a central domain containing 12 almost identical tandem repeats of a 64 amino acid polypeptide. num1-disrupted strains grow normally, but contain many budded cells with two nuclei in the mother cell instead of a single nucleus at the bud neck, while all unbudded cells are uninucleate. This indicates that most G2 nuclei divide in the mother before migrating to the neck, followed by the migration of one of the two daughter nuclei into the bud. Furthermore, haploid num1 strains tend to diploidize during mitosis, and homozygous num1 diploid or tetraploid cells sporulate to form many budded asci with up to eight haploid or diploid spores, respectively, indicating that meiosis starts before nuclear redistribution and cytokinesis. Our data suggest that the NUM1 protein is involved in the interaction of the G2 nucleus with the bud neck.     

Expression of UDP-glucose: poriferasterol glucosyltransferase in the process of differentiation of a true slime mold, Physarum polycephalum

An expression of UDP-glucose:poriferasterol glucosyltransferase activity associated with differentiation of a true slime mold, Physarum polycephalum, from haploid myxoamoebae to diploid plasmodia was demonstrated. In the haploid cells, this enzyme activity was not detected, but after conjugation of the myxoamoebae, the enzyme activity was expressed and increased definitely. In the plasmodial stage, high enzyme activity was maintained constantly. The enzyme was partially purified (35-fold purification, and 28% yield), and molecular weight of 72,000, pH optimum of 7.0, and some characteristics were demonstrated.     

A Genetic Screen for Mutations Affecting Cell Division in the Arabidopsis thaliana Embryo Identifies Seven Loci Required for Cytokinesis

Cytokinesis in plants involves the formation of unique cellular structures such as the phragmoplast and the cell plate, both of which are required to divide the cell after nuclear division. In order to isolate genes that are involved in de novo cell wall formation, we performed a large-scale, microscope-based screen for Arabidopsis mutants that severely impair cytokinesis in the embryo. We recovered 35 mutations that form abnormally enlarged cells with multiple, often polyploid nuclei and incomplete cell walls. These mutants represent seven genes, four of which have previously been implicated in phragmoplast or cell plate function. Mutations in two loci show strongly reduced transmission through the haploid gametophytic generation. Molecular cloning of both corresponding genes reveals that one is represented by hypomorphic alleles of the kinesin-5 gene RADIALLY SWOLLEN 7 (homologous to tobacco kinesin-related protein TKRP125), and that the other gene corresponds to the Arabidopsis FUSED ortholog TWO-IN-ONE (originally identified based on its function in pollen development). No mutations that completely abolish the formation of cross walls in diploid cells were found. Our results support the idea that cytokinesis in the diploid and haploid generations involve similar mechanisms.     

The interrelationship of cell growth and division in haploid and diploid cells of Saccharomyces cerevisiae

The coordination of cell growth and division has been examined in isogenic haploid and diploid strains of Saccharomyces cerevisiae. The average cell volume of the haploid and diploid cells was unaffected by a range of environmental conditions and generation times. For most environments and generation times the mean cell volume of diploid cells was between 1.52 and 1.83 of the haploid cell volume. Both haploid and diploid cell volumes were reduced drastically when the cells were grown in the chemostat with glucose as the limiting substrate. In this environment diploid cells have the same mean cell volume as haploid cells. Diploid cells are more elongated than haploid cells, and the characteristic shape (eccentricity) of the cells is unaffected by all environmental conditions and generation times tested. Mother cell volume increased during the cell cycle, although the pattern of this increase was affected by the environmental conditions. Under most growth conditions detectable mother cell volume increase occurred only during the budding phase, whereas under conditions of carbon limitation detectable increase only occurred during the unbudded phase. A consequence of this result is that the mean cell volume of haploids at bud initiation is relatively constant in all environments, including carbon limitation. This suggests that there is a critical size for bud initiation for haploids which is constant and independent of environmental conditions. The results for diploids are more complex. Coordination of growth and division in haploid cells can be explained by a simple model initially developed for prokaryotes by Donachie. A modification of this model is proposed to account for the results with diploids.     

Breakdown of self/nonself recognition in cannibalistic strains of the predatory slime mold, Dictyostelium caveatum

Dictyostelium caveatum amebas feed upon both bacteria and the amebas of other cellular slime molds. The capacity to feed extensively upon other cellular slime molds is unique to D. caveatum amebas. They are able to phagocytose amebas larger than themselves by nibbling pieces of the cells until they are small enough to ingest. Here we report the isolation from previously cloned stock cultures of stable, cannibalistic strains of D. caveatum in which self/nonself recognition has broken down. Because of the extensive cannibalism, amebas of these strains do not complete multicellular development, and instead wander about for long periods while feeding upon each other. Although the cannibalistic behavior resembles that exhibited by the presumably diploid giant cells in the sexual cycle of other cellular slime molds, these strains are haploid and do not form macrocysts.     

Viability of Physarum polycephalum spores and ploidy of plasmodial nuclei.

Amoebae of Physarum polycephalum carrying the mth mating-type allele may differentiate into plasmodia in the absence of mating. Such plasmodia are haploid and, upon sporulation, produce mainly inviable spores. We have asked whether the viable spores arise from meiotic or mitotic divisions. Using a microfluorometric measurement of the deoxyribonucleic acid content of individual nuclei, we found the fraction of viable spores to be correlated with the proportion of rare, diploid nuclei containing in the generally haploid plasmodium. When homozygous diploid plasmodia were created by heat shocking, spore viability increased dramatically. We suggest that viable spores are produced via meiosis in mth plasmodia, that the mth allele has no effect on sporulation per se, and that the normal source of viable haploid spores is a small fraction of diploid nuclei ubiquitous in haploid plasmodia.     

Involvement of actin dephosphorylation in germination of Physarum sclerotium

The plasmodium of Physarum polycephalum grows without cytokinesis and shows an active cytoplasmic streaming under wet and nutritious conditions. It can undergo reversible differentiation into several types of dormancy to survive in adverse environments. Temperature change or osmotic stress leads to cytoplasmic division of the plasmodium into cells containing one or more nuclei: these form a macrocyst, the spherule. Desiccation also induces cell division of the plasmodium followed by formation of a sclerotium, a dormant body resistant to dry stress. More than half of the actin in a sclerotium is phosphorylated at a single site, threonine 203, resulting in loss of its ability to polymerize into actin filaments. In the present study, actin phosphorylation was found in the sclerotium but not in either the plasmodium or in the spherule. This result suggests that phosphorylation of sclerotium actin may be related to the mechanism associated with desiccation resistance rather than morphological changes through cell compartmentalization in the macrocyst formation. Moreover. dephosphorylation of the phosphorylated form of sclerotium actin began within 5 min after addition of water. Dephosphorylation was not affected by sucrose and sorbitol sugars, but was inhibited by ammonium bicarbonate, ammonium phosphate, sodium phosphate, NaCl, and KCl in a dose-dependent manner. On the other hand, in germination of the sclerotium there was measurable sensitivity to both carbohydrates and salts. Actin dephosphorylation seems to be one of the important processes in the early phase of sclerotium germination.     

SEXUAL REPRODUCTION AND MEIOSIS IN PERIDINIUM INCONSPICUUM LEMMERMANN (DINOPHYCEAE)

In Peridinium inconspicuum Lemmermann, sexual reproduction occurs in both nitrogen-enriched and nitrogen-deficient media. In this homothallic strain, protoplasmic fusion begins between two thecate gametes; but zygote formation is completed in a space outside the fusing pair. This diploid cell can form a plated theca which is shed as the cell enlarges. This spherical zygote then forms a new non-plated theca. The process of ecdysis and the formation of a new non-plated theca is repeated several times. During this process the zygote gradually elongates and by cytoplasmic infurrowing becomes peanut-shaped. Eventually two cells are formed. The first and second meiotic divisions are greatly separated in time. The first meiotic division occurs in the spherical non-thecate zygote. The second meiotic division can occur in the peanut-shaped zygote before it completes cytokinesis. This meiotic division may not be synchronous, occasionally resulting in a trinucleate stage. Eventually four flagellated, haploid products are produced.     

Chemotaxis toward carbohydrates and amino acids in Physarum polycephalum

A method is described for assaying chemotaxis in the acellular slime mold Physarum polycephalum. It consists of measuring the amount of plasmodium that moves on a strip of nitrocellulose membrane filter Millipore in response to a gradient of an attractant. Time course of chemotactic response of the slime mold is described. Different factors that affect chemotaxis in the slime mold such as: culture care and stage of growth of microplasmodia, substratum used for cell movement, nature of the gradient, effect of salts, pH and temperature are described. From concentration-response curves for different attractants several parameters of the chemotactic effect, such as threshold concentration, half maximal concentration, and maximal effective concentration can be determined. As a group, sugars are more effective chemotactic agents than amino acids. Glucose and galactose, which support the growth of the slime mold, are shown to have high positive chemotactic effect. 3-O-Methyl- -glucose and 2-deoxy- -glucose are two sugars that do not support growth but are very effective attractants. Conversely, fructose which supports slime mold growth is at best a weak attractant. The results support the view that the chemotactic effects of different sugars are not dependent on their growth-supporting value.     

Achene morphology and slime structure in some taxa of Artemisia L. and Neopallasia L. (Asteraceae)

We have examined slime cell distribution on the surface of the achenes of some Artemisia and Neopallasia taxa, as well as slime composition, envelope formation during the hydration, and slime relation to different morphological features and environmental factors. The results of the studies show a characteristic pattern of slime cells distribution, which could differ between taxa. The slime in the taxa studied belongs to the cellulose type and consists of two components i.e., pectins and cellulose. Although all fruits contain slime cells, not all of them show the slime envelope formation. Plants occurring in dry habitats (such as A. barrelieri) or annual species (such as A. annua) are characterised by a large amount of slime and a fast process of slime envelope formation. Slime production has not been observed in some polyploid populations (A. campestris and A. campestris ssp. sericea) and in two species occurring in relatively fertile habitats (A. verlotiorum, A. vulgaris). A reason for this may be either the immaturity of polyploid fruits leading to the production of a scarce, not detectable slime amount or, alternatively, the occurrence of not functional slime cells. Slime facilitates and stimulates the germination, as well as the adherence of the fruits to the ground or to animals (for dispersal). The slime could play important role in the distribution and colonisation of new habitats in many Artemisia taxa.     

Proteome comparative analysis of gynogenetic haploid and diploid embryos of goldfish (Carassius auratus)

Recently, it was found that in the gynogenetic haploid and diploid embryos of goldfish, which have exactly the same genome, the haploid condition results in obstruction of gene expression and abnormal development while the diploid embryos have normal gene expression and development. A diploid-dependent regulatory apparatus was proposed to regulate gene expression. To study the difference at the protein expression level of the embryos of haploid and diploid in development, we extracted the total proteins of both the gynogenetic haploid and diploid embryos of goldfish in the same eye formation stage. Two-dimensional polyacrylamide gel electrophoresis was used to separate proteins. The stained gel images were analyzed with the PDQUEST software. A part of protein spots that were differentially expressed in haploid and diploid embryos were identified by matrix assisted laser desorption/ionisation-time of flight-mass spectrometry and database analysis. Sixteen protein spots that were absolutely different (only expressed in diploid embryos but not in haploid embryos or vice versa) and 16 protein spots that were up- and downregulated were identified unambiguously, which include some proteins that are correlative with eyes development, nerve development, developing regulation, cell differentiation, and signal transduction. The different significantly gene expression during embryos developing between diploid and haploid is demonstrated.