Jennifer Dee

Plasmodium formation without change in nuclear DNA content in Physarum polycephalum.

The Colonia isolate of Physarum polycephalum produces plasmodia within amoebal clones. Wheals demonstrated genetically that amoebae of the C50 strain of this isolate, when crossed with heterothallic amoebae, yielded recombinant progeny. He concluded that nuclear fusion and meiosis occurred in these crosses and suggested that nuclear fusion was also involved in plasmodia formation in clones. He thus designated the strain ‘homothallic’. In the present work genetic evidence is presented which indicates that the Colonia strain CL , when crossed with heterothallic strains, also yields recombinant progeny and thus undergoes nuclear fusion and meiosis. Microdensitometric measurements of nuclear DNA content are reported which indicate that CL amoebae are haploid like heterothallic amoebae, and crossed plasmodia are diploid. However, clonally formed CL plasmodia were found to have the same G 2 nuclear DNA content as CL amoebae. This observation excludes the possibility of nuclear fusion when plasmodia form within clones of CL amoebae and therefore the strain cannot be homothallic. Two alternatives, apogamy and coalescence, are proposed as the most likely mechanisms for clonal plasmodium formation in strain CL .

Defined and semi-defined media for the growth of amoebae of Physarum polycephalum.

Amoebae of the true slime mould Physarum polycephalum were cultured in two fully-defined liquid media containing amino acids, glucose, three vitamins and a buffered salts solution. Absolute requirements were demonstrated for methionine, haematin, thiamine and biotin, all of which were known to be specific requirements of the plasmodial stage. Methods are described for large-scale culture in three semi-defined media.

A gene conferring actidione resistance and abnormal morphology on Physarum polycephalum plasmodia

Plasmodia of P. polycephalum homozygous and heterozygous for a mutation ( act ) which confers resistance to actidione on the haploid amoebal stage have been tested for resistance to actidione in liquid semidefined medium. Growth of homozygous resistant ( act/act ) plasmodia determined by protein or pigment assay was inhibited less by actidione than growth of homozygous-sensitive or heterozygous plasmodia. The mutant ( act ) is therefore recessive but, contrary to a previous report, confers resistance on homozygous plasmodia. The same mutation affects the morphology of homozygous ( act/act ) plasmodia on agar medium, causing slow growth, fragmentation and excessive sliminess.

Apogamic development of plasmodia in the myxomycetePhysarum polycephalum: a cinematographic analysis

SummaryStrain CL ofPhysarum polycephalum forms multinucleate plasmodia within clones of uninucleate amoebae. The plasmodia have the same nuclear DNA content as the amoebae. Analysis of plasmodial development, using time-lapse cinematography, showed that binucleate cells were formed as a result of nuclear division in uninucleate cells. Binucleate cells developed into plasmodia by further nuclear divisions and cell fusions. No fusions involving uninucleate cells were observed. A temporary increase in cell and nuclear size occurred at the time of binucleate cell formation.

Genetic Factors Determining the Growth of Physarum polycephalum Amoebae in Axenic Medium

Summary: Evidence is presented that in the true slime mould Physarum polycephalum the ability of the amoebal strain CLd-AXE to grow in axenic medium is determined by mutations in two genes axeA and axeB and that the axenic growth of RSD4-AXE amoebae is also under genetic control. Mutant amoebal strains are also able to grow on bacterial lawns and the ability to grow in axenic media persists during prolonged culture on bacteria. However, some of the mutant strains grow less well on bacteria than strains of similar genetic background which are unable to grow in axenic media. CLd-AXE has the same nuclear DNA content (0.59 pg per nucleus) as CLd, the strain from which it was derived. Amoebae able to grow in axenic media were also derived from strains E27 and LU858 which carry mutations for temperature sensitivity and leucine requirement expressed in the plasmodial phase. Tests in axenic media showed that these mutations were expressed in the amoebal phase. The elucidation of the genetic basis of axenic growth will allow the construction of a range of amoebal strains able to grow in axenic media and this will greatly facilitate the isolation and analysis of mutants in this organism.

Isolation and Analysis of Amoebal–Plasmodial Transition Mutants in the Myxomycete Physarum polycephalum

Plasmodium formation in the Myxomycete Physarum polycephalum normally involves fusion of haploid amoebae, carrying different alleles at the mating type (mt) locus, to give diploid plasmodia. Strains carrying the mth allele are capable of undergoing the amoebal-plasmodial transition with high efficiency within amoebal clones, resulting in the formation of haploid plasmodia. NMG mutagenesis of mth amoebae, followed by an enrichment procedure, was used to isolate mutants in which such clonal plasmodium formation was either delayed or absent. Thirteen mutants of the second type were analysed. Three of these were temperature-sensitive for plasmodium formation. All thirteen mutants were able to form diploid crossed plasmodia when mixed with a mtx strain. Three new genes were identified and designated npfA, npfB and npfC. A mutant allele of npfA rendered clonal plasmodium formation temperature-sensitive, but did not prevent crossing at the non-permissive temperature with derived strains carrying the same mutant allele. No recombination was detected between npfB or npfC and mt, but npfA was unlinked to mt and a locus (apt-1) shown in a previous study to be involved in plasmodium formation. The genes npfB and npfC were distinguished by complementation analysis. Strains of the genotype npfB~; npfC behaved in the same way as strains carrying the mt2 allele. The nature of the mutants and the role of the mating-type locus in the initiation of plasmodium formation are discussed.

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.

The molecular biology of Physarum polycephalum

wilhelm Stockem and Jorg Kukulies Institute of Cytology University of Bonn Bonn, FRG Tetramethylrhodaminyl (TRITC)-phalloidin and isolated muscle or Physarum G-actin labeled with various fluorochromes were microinjected into living stages of Physarum polycephalum (cell fragments and microplasmodia). Subsequent analysis of the intracellular redistribution of the molecular probes by fluorescence microscopy, video-enhancement, and digital image processing revealed that polymerization-depolymerization and contraction-relaxation cycles of the microfilament system are functionally related to changes in cell shape, protoplasmic streaming activity, and ultrastructural morphology of the specimens. In relaxed cell fragments, TRITCphalloidin and rhodamine-isothiocyanate (RITC) -actin first diffuse randomly and then are locally incorporated into a thin cortical layer at the internal face of the plasma membrane. During Ca2+induced contraction, the fluorescent layer starts to detach from the plasma membrane, thus causing separation of the central granuloplasm from the peripheral hyaloplasm. Thin sections of both relaxed and contracted specimens demonstrate that the fluorescent layer in living cell fragments coincides exactly with a sheath of more or less oriented microfilaments. In contrast, RITC-bovine serum albumin injected as a control is excluded from those regions that show intense fluorescence with RITC-actin and TRITC-phalloidin and the presence of an actin network b¥ electron microscopy.

Growth and development in relation to the cell cycle inPhysarum polycephalum

SummaryIn 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.

Analysis of development and growth in a mutant of Physarum polycephalum with defective cytokinesis

Abstract Cultures of amoebae of the mutant strain ATS23 isolated from strain CLd of Physarum polycephalum contain multinucleate cells and cells with increased nuclear DNA content. Plasmodia derived from ATS23 clones show abnormal morphology and defective sporulation. All abnormalities are enhanced by high incubation temperature (31 °C). Genetic analysis suggested that all the abnormalities were caused by a single mutation, denoted hts-23 . The kinetics of plasmodium formation were followed in cultures of apogamic amoebae carrying hts-23 and hts + (wild type) respectively. Results indicated that, relative to wild type, hts-23 did not increase the rate of plasmodium formation. There was evidence that, in both mutant and wild-type strains, commitment to plasmodium development occurred in uninucleate cells. Analysis of cell pedigrees by time-lapse cinematography indicated that the primary abnormal event in cultures of hts-23 amoebae was failure of cytokinesis; an apparently complete cleavage furrow was formed but cell separation failed, resulting in a binucleate cell. This event occurred randomly in pedigrees in which the majority of divisions were completed normally; its frequency increased during incubation at 31 °C. All other abnormalities in hts-23 amoebal cultures could be attributed to this primary event, assuming that DNA synthesis continued in the absence of cytokinesis and that the binucleate cells underwent the amoebal type of “open” mitosis, allowing the possibility of spindle fusion. This implies that the acquisition of “closed” mitosis is an essential early step in plasmodium development.