4D confocal microscopy of Dictyostelium discoideum morphogenesis and its presentation on the Internet

 Methods to present three-dimensional (3D) and time series of 3D datasets (4D) are demonstrated using the recent advances in confocal microscopy and computer visualization. The process of cell sorting during tip formation in the slime mould Dictyostelium discoideum is examined as an example by in vivo confocal microscopy of spectrally different green fluorescent protein (GFP) variants as reporters of cell-type specific gene expression. Also, cell sorting of the co-aggregating slime mould species D. discoideum and D. mucoroides is observed using a GFP variant and a spectrally distinguishable fluorescent vital stain. The confocal data are handled as 3D and 4D datasets, their processing and the advantages of different methods of visualization are discussed step by step. Selected sequences of the experiments can be viewed on the Internet, giving a much better impression of the complex cellular movements during Dictyostelium morphogenesis than printed photographs. Received: 17 February 1998 / Accepted: 14 June 1998     

INFLUENCE OF IONIC CONDITIONS ON CELL DIFFERENTIATION AND MORPHOGENESIS OF THE CELLULAR SLIME MOLDS

Effects of various ionic conditions on the development of the cellular slime molds D. discoideum and D. mucoroides were studied. A certain concentration of lithium ions (7 mM) promoted differentiation of the stalk cells and conversely inhibited formation of the spores. The presence of calcium and magnesium ions was needed for Li to manifest its specific effect. A high concentration of Ca (100-120 mM) also facilitated differentiation of the stalk cells. On the other hand, fluoride ions stimulated considerable formation of spores at 15 mM. In the absence of divalent cations, sodium ions inhibited morphogenesis and cell differentiation proportionately with its concentration, and complete inhibition was obtained at 20 mM. The inhibitory effect of Na was nullified by addition of small amounts of Ca. Possible mechanisms by which these ions exert their influences on development of this organism were discussed.     

Detection and Measurement of Staphylococcus epidermidis Slime Using an ELISA Technique

A polyclonal rabbit anti-serum against the strong slime-producing Staphylococcus epidermidis strain RP62A was absorbed with the slime-negative phase variant of this strain PV1 in order to remove not slime-specific antibodies. Using this antiserum we established an ELISA which enables detection of slime production in S. epidermidis extracts. The ELISA showed high absorbance when extracts from slime-positive strains (confirmed in the tissue culture tube test) were used as antigens. The high absorbance of slime-positive strains was greatly reduced by periodate oxidation of the extracts and was resistant to proteinase digestion suggesting that the detected antigen is composed of polysaccharides. In contrast to other rapid and simple laboratory detection methods for S. epidermidis slime, the slime-specific ELISA gave positive results in the presence of human serum.     

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.     

THE OCCURRENCE AND DISTRIBUTION OF ACRASIEAE IN FOREST SOILS. I. EUROPE

The deciduous forests of Europe contain fewer species of cellular slime molds than those of Eastern North America. Diclyostelium mucoroides is the most widely distributed and the most abundant species. In Europe this species is as a rule the sole dominant species in a forest-soil acrasian population, a situation that rarely occurs in North America. An explanation of this difference may lie in the severity of the Pleistocene climate on European forests which resulted in the elimination of suitable habitats for the Acrasieae over a long period of time, and the replacement of the original forest by one much poorer in tree species. It is suggested that this modern forest does not offer the diverse of habitat which may be requited to support a mixed population of cellular slime molds.     

Slime Production by Pseudomonas aeruginosa

Adequate experimental conditions for slime production by Pseudomonas aeruginosa were investigated using a cellophane plate method. Definite slime production was observed on heart infusion agar, brain heart infusion agar, yeast extract agar and synthetic agar, but not on nutrient agar. The addition of phosphate to the nutrient agar above 0.05% caused visible slime formation. Incubation at 37 C resulted in a higher yield of slime than at 25 C. Longer incubation seemed more favorable for slime production, while the pH reaction of the test media did not effect the slime yield. All the test cultures of P. aeruginosa produced large amounts of slime by this procedure. Cultures of Pseudomonas fluorescens, other Pseudomonas spp. and certain vibrios also produced slime under these experimental conditions.     

In vitro screening of taxol,an anticancer drug produced by the fungus,Colletotrichum capsici

The fungus Colletotrichum capsici was isolated from the diseased fruits of Chilli plant, Capsicum annuum. The isolated test fungus was identified by its morphological and molecular characteristic features. For the first time, the fungus was screened for the production of taxol on modified liquid medium. The presence of taxol was confirmed by the spectroscopic and chromatographic methods of analyses. The amount of taxol produced by this fungus was quantified by HPLC. The maximum amount of fungal taxol production was recorded as 687 μg/L. The production rate was 13 740‐fold higher than that, previously reported for the fungus Taxomyces andreanae. The extracted fungal taxol showed a strong cytotoxic activity in an in vitro culture of human cancer cells indicating that the increase in taxol concentration induces increased cell death. A PCR‐based screening for taxadiene synthase (ts), a unique gene in the formation of the taxane skeleton, confirmed the molecular blueprint for taxol biosynthesis. The results show that the fungus C. capsici is an excellent candidate for an alternate source of taxol supply and can serve as a potential species for genetic engineering to enhance the production of taxol to a higher level.     

THE DISTRIBUTION OF DICTYOSTELID CELLULAR SLIME MOLDS IN SOUTHERN CALIFORNIA WITH TAXONOMIC NOTES ON SELECTED SPECIES

The occurrence and distribution of Dictyostelid cellular slime molds in southern California were investigated by means of a clonal isolation technique that permitted quantitative sampling of populations. Thirteen species were isolated. These include (in approximate order of decreasing frequency): Dictyostelium rosarium Raper and Cavender, D. mucoroides Brefeld, D. sphaerocephalum (Oud.) Sacc. and March., D. aureum Olive, Polysphondylium pallidum Olive, D. minutum Raper, D. giganteum Singh, D. purpureum Olive, P. violaceum Brefeld, D. mucoroides variant, Acytostelium leptosomum Raper and Quinlan, D. polycephalum Raper, and a new species of Dictyostelium. D. rosarium and D. aureum, both rarely reported in the literature, were common and widespread. Trends in habitat preference of the Dictyostelids encountered are discussed. Selected taxonomic details are provided for D. rosarium, D. aureum and members of the “D. mucoroides complex“—D. mucoroides, D. sphaerocephalum, D. giganteum, and a D. mucoroides variant.     

THE YELLOW-PIGMENTED DICTYOSTELIA

The cellular slime molds described herein are of a clear golden yellow color and belong to two naturally occurring groups. One we call Dictyostelium mexicanum sp. n. because it is found in soils from various parts of tropical and subtropical Mexico. The well-tapered stalk and disklike base are somewhat reminiscent of D. discoideum. The other is regarded as D. aureum E. W. Olive (1901) with which we feel it is closely identified if not identical. In the latter group the form of both the aggregation and sorocarp is suggestive of D. mucoroides. Some less pigmented isolates are separated as D. aureum var. luteolum var. n.     

Genetic transactivation of Dissociation elements in transgenic tomato plants

Summary A DNA amplification is correlated with the dominant, unstable cob-354 cobalt resistance trait in the cellular slime mold, Dictyostelium discoideum. The amplified DNA is present as about 50 copies of an extrachromosomal element. Cells grown under nonselective conditions in the absence of cobalt ions lose both the cobalt resistance trait and all extrachromosomal copies of the amplified DNA. The amplified DNA is transferrable to new genetic backgrounds by parasexual genetic crosses. These results explain the inability to map the cob-354 trait to a linkage group. The chromosomal origin of the amplified DNA is group III or VI. Thus the resistance trait appears to be independent of the previously known cobalt resistance locus, cobA, which maps to group VII. A developmental defect involving the production of multiply-tipped aggregates that do not complete fruiting body formation also is correlated with the presence of the amplified DNA.     

Nuclear actin bundles in Amoeba, dictyostelium and human HeLa cells induced by dimethyl sulfoxide

In a previous study we demonstrated that dimethyl sulfoxide (DMSO) induces the formation of microfilament bundles in the interphase nucleus of a cellular slime mold, Dictyostelium mucoroides [12], in which the microfilaments bound rabbit skeletal muscle heavy meromyosin, forming an ‘arrowhead’ structure, and that this binding could be reversed by Mg²⁺ and ATP. In the present study, we show electron microscopic data demonstrating the occurrence of such microfilament bundles in the nucleus of Amoeba proteus and human HeLa cells, as well as in D. mucoroides. The similarities in the morphology and dimension of the microfilanets, as well as the specific conditions by which they are induced, suggested that these microfilaments are actin. We present evidence that actin is involved in interphase nucleus of a variety of organisms, and that DMSO acts on the molecules to induce microfilament bundles specifically in the nucleus.     

THE GENUS PROTOSTELIUM

Olive , L. S. (Columbia U., New York, N. Y.) The genus Protostelium . Amer. Jour. Bot. 49(3): 297–303. Illus. 1962.—Two new species of this simplest genus of the cellular slime molds are described: P. fimicola , a species with spherical spores and gelatinizing stalks occurring on dung, and P. arachispora , an elongate-spored species from dead plant material. The first has been found in North Carolina and the West Indies, while the second species has been found only once in North Carolina. Protostelium mycophaga var. major , from Connecticut, has distinctly larger spores than the original species. Further data on the distribution and development of P. mycophaga are presented. The genus is transferred to a family of its own, the Protosteliaceae .     

Fruit structure of 12 Turkish endemic <Emphasis Type="Italic">Tripleurospermum</Emphasis> Sch. Bip. (Asteraceae) taxa and its taxonomic implications

This study concerns the evaluation of micromorphological and anatomical characters of fruit (achene-cypsela) in 12 Turkish endemic Tripleurospermum taxa using multivariate analyses (cluster analysis, principal component analysis, one-way analysis of variance). Pericarp in all taxa examined is mainly composed of several layers of parenchymatous and sclerenchymatous cells with one vascular bundle in each rib. In the achene, the thickness and width of lateral and adaxial ribs with presence or absence of a slime envelope have high taxonomic value for Tripleurospermum at interspecific levels. The slime envelope formation is also correlated with ploidy levels and habitats of some taxa in Tripleurospermum.     

Chitosomes from the wall-less “slime” mutant of Neurospora crassa

Cell-free extracts from the wall-less slime mutant of Neurospora crassa and the mycelium of wild type exhibit similar chitin synthetase properties in specific activity, zymogenicity and a preferential intracellular localization of chitosomes. The yield of chitosomal chitin synthetase from sline cells was essentially the same irrespective of cell breakage procedure (osmotic lysis or ballistic disruption) —an indication that chitosomes are not fragments of larger membranes produced by harsh (ballistic) disruption procedures. The plasma membrane fraction, isolated from slime cells treated with concanavalin A, contained only a minute portion of the total chitin synthetase of the fungus. Most of the activity was in the cytoplasmic fraction; isopycnic sedimentation of this fraction on a sucrose gradient yielded a sharp band of chitosomes with a buoyant density=1.125 g/ cm³. Approximately 76% of the total chitin synthetase activity of the slime mutant was recovered in the chitosome band. Because of their low density, chitosomes could be cleanly separated from the rest of the membranous organelles of the fungus. Apparently, the lack of a cell wall in the slime mutant is not due to the absence of either chitosomes or zymogenic chitin synthetase.Abbreviations Con A concanavalin A - d buoyant density in g/cm³ - GlcNAc N-acetyl-D-glucosamine - MES 2-[N-morpholino]ethanesulfonic acid - UDP-GlcNAc uridine diphosphate N-acetyl-D-glucosamine     

Genetic, biochemical, and developmental studies of nystatin resistant mutants in Dictyostelium discoideum

Summary Conditions are given for the isolation of nystatin resistant mutants of the cellular slime mold Dictyostelium discoideum. These mutants fall into three phenotypic groups; corresponding to three genes: nysA, nysB, and nysC. Mutants in nysB and nysC affect sterol metabolism since they have altered sterol compositions. Each group contains several unique, but as yet unidentified, sterols in place of the wild type sterol. The nysC strains are most nystatin resistant, display altered sensitivity to some drugs, and grow on nystatin from amoebae or spores. All other mutants are nystatin resistant only as amoebae. Although nysC mutants grow normally, they make small fruiting bodies which appear to result from the formation of smaller aggregates.Supported by N.I.H. grant GM 18476     

THE OCCURRENCE AND DISTRIBUTION OF ACRASIEAE IN FORESTS OF SUBTROPICAL AND TROPICAL AMERICA

Forests of the subtropical and tropical regions of North America harbor cellular slime molds not found in the soils of temperate deciduous forests investigated previously. However, most species found in the temperate forest are common in the tropics. Although the diversity of forms is greater in the soils of tropical forests the numbers of Acrasieae per unit of soil are comparable. Characteristic of tropical and subtropical forest soils are Acrasieae bearing crampon bases, of which four new species of Dictyostelium are presently known. Also present, but less frequently isolated, are two other new species of the genus Dictyostelium and two still undescribed species of the Guttulinaceae. Occasional isolates of D. purpureum and D. discoideum were found that produce macrocysts, which seem, also, to be confined to tropical and subtropical areas. Macro-cysts were previously known only in D. mucoroides and D. minutum isolated from temperate forest soils. The occurrence and distribution of Acrasieae in warm temperate desert and mesquite-scrub, in subtropical hammock, and in tropical thorn, deciduous, seasonal evergreen, rain, and cloud forests were investigated. Acrasieae were well represented in all of these forests except desert. The number of species and the total populations were largest in seasonal evergreen forests. The composition of the cellular slime mold populations and the dominant species within these populations could be related to the soil environment as expressed by the dominant vegetation.     

SEPTAL PORES IN THE HETEROBASIDIOMYCETIDAE,PUCCINIA GRAMINIS AND P. RECONDITA

The septal pores in uredial mycelium of Puccinia graminis and P. recondita lack the septal swelling and septal pore cap (dolipore-parenthosome configuration) typically associated with the pores of previously investigated Homobasidiomycetidae and the Tremellales among the Heterobasidiomycetidae. The pores in young hyphae of these two species of Puccinia are characterized by the presence of a cytoplasmic matrix which apparently occludes the pore and acts as a plug, thus preventing the migration of organelles from cell to cell. Large vesicles are typically present at the periphery of the pore matrix and the matrix may be very incompletely bounded by a membrane. Nuclei and other cytoplasmic structures migrate from cell to cell through an opening in the septum lateral to the pore. The available evidence indicates that this peripheral gap in the septum results from a breakdown of a portion of an initially complete septum rather than from incomplete septum formation. In addition to the centripetally formed septa, the hyphae of P. graminis and P. recondita are further compartmentalized by shallow infoldings of the lateral wall and limited unilateral septum formation. There is apparent free passage of cellular material between adjacent compartments.     

Nondegradative pisatin-resistance inDictyostelium discoideum,Neurospora crassa andNectria haematococca: Similarities and differences

Paradoxically, on pisatin-medium (150 μg/ml) the cellular slime mouldDictyostelium discoideum grows only when plated as spores but not when plated as amoebae. The recent discovery of inducible nondegradativc pisatin resistance in amoebae has allowed us to formulate a model that resolves this paradox. In this model, the germinating amoeba is postulated to acquire a pisatin-resistance phenotype while ensconced within the spore wall. This article reviews the findings on which this model is based and extends it to also account for the differences in pisatin sensitivity phenotype that result from sterol alteration in cellular slime mould^s and fungi.     

Fruiting-body formation, cultivation properties, and host specificity of a fungicolous fungus, Asterophora lycoperdoides

We report the fruiting-body formation and cultivation properties of Asterophora lycoperdoides, a fungicolous fungus. Asterophora lycoperdoides formed fruiting bodies on potato dextrose agar medium in approximately 1 week, although this fungus shows high host specificity to Russula nigricans in nature. Optimal temperature of mycelial growth and fruiting-body formation was 25°C. Mannitol or soluble starch was preferably used as a carbon source, and amino nitrogen was preferably used as the nitrogen source. For a better understanding of the relationship between A. lycoperdoides and R. nigricans, we cultivated A. lycoperdoides on media supplemented with freeze-dried fruiting bodies of various fungi. The germination rate was approximately 2.5 times higher on the medium containing freeze-dried R. nigricans than that on the PDA medium. The mycelia extended most rapidly in the presence of R. nigricans. Furthermore, the stipe length of its fruiting body was the longest on the medium containing R. nigricans. These results indicated that A. lycoperdoides can grow faster by utilizing certain substances that are abundantly contained in R. nigricans, such as mannitol, or by utilizing R. nigricans itself. It is considered that the constituents of R. nigricans might contribute to the host specificity of A. lycoperdoides.