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     

Indirect immunofluorescent staining of the microtubules in interphase and mitotic amoebae of the myxomycetePhysarum polycephalum

Summary The microtubules ofPhysarum amoebae have been decorated with rat antibodies against yeast tubulin. The indirect fluorescent staining observed in interphase amoebae and in flagellated amoebae is consistent with the three-dimensional reconstructions previously deduced from electron microscopic studies. Mitotic amoebae exhibit a pattern of fluorescence which is similar to that exhibited by mammalian cells and is consistent with the previous electron microscopic studies, except that we also observe pole-pole microtubule fibers during metaphase and anaphase and the presence of a typical midbody during cytokinesis. The various types of tripolar mitosis which are observed suggest that there is a regulatory mechanism allowing the formation of pseudo-bipolar mitotic apparatuses in amoebae possessing more than two mitotic centers during mitosis. The mitotic center, located in the middle of the centrosphere, is not fluorescent after staining of the monoasters induced with taxol suggesting the absence of tubulin in the mitotic center.     

Trophic Controls on Stage Transformations of a Toxic Ambush-Predator Dinoflagellate1

ABSTRACT. The toxic dinoflagellate, Pfiesteria piscicida, was recently implicated as the causative agent for about 50% of the major fish kills occurring over a three-year period in the Albemarle-Pamlico Estuarine System of the southeastern USA. Transformations between life-history stages of this dinoflagellate are controlled by the availability of fresh fish secretions or fish tissues, and secondarily influenced by the availability of alternate prey including bacteria, algae, microfauna, and mammalian tissues. Toxic zoospores of P. piscicida subdue fish by excreting lethal neurotoxins that narcotize the prey, disrupt its osmoregulatory system, and attack its nervous system. While prey are dying, the zoospores feed upon bits of fish tissue and complete the sexual phase of the dinoflagellate life cycle. Other stages in the complex life cycle of P. piscidia include cryptic forms of filose, rhizopodial, and lobose amoebae that can form within minutes from toxic zoospores, gametes, or planozygotes. These cryptic amoebae feed upon fish carcasses and other prey and, thus far, have proven less vulnerable to microbial predators than flagellated life-history stages. Lobose amoebae that develop from toxic zoospores and planozygotes during colder periods have also shown ambush behavior toward live fish. In the presence of abundant flagellated algal prey, amoeboid stages produce nontoxic zoospores that can become toxic and form gametes when they detect what is presumed to be a threshold level of a stimulatory substance(s) derived from live fish. The diverse amoeboid stages of this fish “ambush-predator” and at least one other Pfiesteria-like species are ubiquitous and abundant in brackish waters along the western Atlantic and Gulf Coasts, indicating a need to re-evaluate the role of dinoflagellates in the microbial food webs of turbid nutrient-enriched estuaries.     

Bau und Funktion des Süßwasserschwamms Ephydatia fluviatilis L. (Porifera)

Zusammenfassung Die Kragengeißelkammern von Ephydatia fluviatilis entstehen frei im Mesenchym. An den Entstehungsorten trifft man auf Anhäufungen rundlicher Zellen, die allem Anschein nach von Archäocyten stammen, jedoch kleiner sind als diese und einen nukleoluslosen Kern besitzen. Hierbei handelt es sich um Choanoblasten, die zunächst eine Geißel, später den Kragen ausbilden und sich als Choanocyten zu Kragengeißelkammern zusammenfügen.Die im Mesenchym vorläufig fertiggestellten Kragengeißelkammern gelangen an das Endopinacocytenepithel des ausführenden Kanalysystems. Daraufhin bilden sich die tangierten Choanocyten zu Konuszellen um. Das Endopinacocytenepithel antwortet seinerseits mit der Ausbildung einer Poruszelle pro Kragengeißelkammer. Die Porocyten gehen mittels der konfrontierten Konuszellen dauerhafte Verbindungen mit den zugehörigen, nunmehr funktionstüchtigen Kragengeißelkammern ein.

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Structure and function of the fresh-water sponge Ephydatia fluviatilis L. (Porifera)VIII. The origin and development of the flagellated Chambers and their junction with the excurrent canal system

Summary The flagellated chambers of Ephydatia fluviatilis arise at scattered sites within the mesenchyme. Each such site is marked by an accumulation of rounded cells, which appear to be derived from archaeocytes in most respects except that they are smaller than the latter and have no nucleoli in the nucleus. These are choanoblasts, which first develop a flagellum and later a collar; eventually, as choanocytes, they become arranged so as to form a flagellated chamber.Having reached this preliminary stage of completion in the mesenchyme, the flagellated chambers migrate to the endopinacocyte epithelium of the excurrent canal system. Then the choanocytes at the contact point are converted to cone cells. The endopinacocyte epithelium in turn responds by developing one pore cell for each flagellated chamber. The porocytes become permanently joined to the chamber by way of the adjacent cone cells, and from this time on the flagellated chamber is functional.

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Abkürzungen A Archäocyte - aK ausführender Kanal - B Bakterien - Ch Choanocyte - eK einführender Kanal - G Geißel - GK Kragengeißelkammer - GK-A Anlage von Kragengeißelkammern - K Zellkern - Kr Kragen - KZ Konuszelle - M Mesenchym - N SiO₂-Nadel - PC Endopinacocyten - PD Pinacoderm - PV pulsierende Vakuole - PZ Porenzelle - S Gemmulaschale - Sk Skleroblast - Sp Spongin - SR Subdermalraum     

Events in the amoebal-plasmodial transition ofPhysarum polycephalum studied by enrichment for committed cells

Summary Amoebae of strain CLof Physarum polycephalum undergo apogamic development to form multinucleate plasmodia. During the amoebalplasmodial transition, large uninucleate cells become irreversibly committed to plasmodium development. In developing cultures, amoebae lose the ability to flagellate before they become committed. Enriched suspensions of committed cells can be obtained by inducing asynchronous differentiating cultures to flagellate and passing the cells through a glass bead column. Committed cells can be cultured to form plasmodia on bacterial lawns or in axenic liquid medium but cannot be cultured on axenic agar medium. Uninucleate committed cells express tubulin isotypes characteristic of amoebae, but after culture in axenic liquid medium, the cells express plasmodial specific tubulin isotypes.Abbrevations SDM Semi-defined medium - DSDM Dilute semidefined medium - LIA Liver infusion agar - SBS Standard bacterial suspension - IEF Isoelectric focussing - SDS Sodium dodecyl sulphate - PAUF Precommitted amoebae unable to flagellate (for the explanation of these cells see text).     

Polypeptide compositions of amoebae of the cellular slime mouldDictyostelium discoideum separated by partitioning during development

Amoebae of the cellular slime mouldDictyostelium discoideum at 8 h or l0 h development were separated into two populations by countercurrent distribution in a dextran-poly(ethylene glycol), two-phase system. Two-dimensional, polyacrylamide-gel electrophoresis was then used to separate the polypeptides from the populations of amoebae. The two populations of amoebae at 8 h development differed sn polypeptide composition as did the populations separated at 10 h development. This confirms that cell differentiation is initated inD. discoideum prior to g h development.     

Abundance of Naked Amoebae in Sediments of Hiroshima Bay, Seto Inland Sea of Japan

ABSTRACT The present paper provides the first data on naked amoebae from sediments of Hiroshima Bay. Three stations in the inner part of the bay were sampled over a three-month period. Abundance of naked amoebae ranged from 1,019 to 45,561 cells/g dry sediment. Results indicate: (i) surface sediment populations in most cases were higher than subsurface populations; (ii) there was some evidence of temporal variation with counts generally increasing from March to May: and (iii) the site located near Hiroshima City had fewer amoebae on several occasions than the other two sites. There was a negative exponential relationship between acid-volatile sulfide concentration and abundance of amoebae. Most amoebae were small with the average size ranging from 6.6–14 μm. Morphotype 1, amoebae that extend lobose pseudopodia or subpseudopodia during normal locomotion, were dominant (40–100% of enumerated amoebae). Morphotypes 2 and 3 (limax amoebae) were found in lower numbers than the other two morphotypes. The proportion of amoebae occupied by Morphotype 4 (fan-shaped or discoidal-flattened amoebae) was higher at a lower total abundance.     

Cell-cycle regulation of center initiation in Dictyostelium discoideum

The center-initiating behavior of Dictyostelium discoideum amoebae in various cell-cycle phases was investigated. Small populations of synchronized AX-2 cells were seeded 1 in 1000 into cultures of a nonsignaling mutant (NP160) incapable of initiating centers. The ability of the wild-type AX-2 cells to initiate centers among mutant amoebae displayed cell-cycle regulation. Approximately 50% of a population of S-phase cells initiated centers while only 7.5% of a population of late G2-phase cells resulted in center formation. The timing of center formation also varied with cycle position. Synchronous cultures containing only AX-2 S-phase amoebae (no NP160) displayed the initial signs of aggregation after 4.5 hr of starvation and streaming into the aggregate was complete after 6 hr. In contrast, cultures of late G2-phase amoebae initiated aggregation centers after 5.5 hr of starvation and did not complete streaming until 7.5 hr. In addition, the number of aggregates formed by these synchronous cultures of AX-2 cells also varied with cycle position. In general, these results suggest a cell-cycle modulation of the autonomous signaling responsible for center initiation.     

Three‐dimensional fate mapping of larval tissues through metamorphosis in the glass sponge Oopsacas minuta

The tissue of glass sponges (Class Hexactinellida) is unique among metazoans in being largely syncytial, a state that arises during early embryogenesis when blastomeres fuse. In addition, hexactinellids are one of only two poriferan groups that already have clearly formed flagellated chambers as larvae. The fate of the larval chambers and of other tissues during metamorphosis is unknown. One species of hexactinellid, Oopsacas minuta, is found in submarine caves in the Mediterranean and is reproductive year round, which facilitates developmental studies; however, describing metamorphosis has been a challenge because the syncytial nature of the tissue makes it difficult to trace the fates using conventional cell tracking markers. We used three‐dimensional models to map the fate of larval tissues of O. minuta through metamorphosis and provide the first detailed account of larval tissue reorganization at metamorphosis of a glass sponge larva. Larvae settle on their anterior swimming pole or on one side. The multiciliated cells that formed a belt around the larva are discarded during the first stage of metamorphosis. We found that larval flagellated chambers are retained throughout metamorphosis and become the kernels of the first pumping chambers of the juvenile sponge. As larvae of O. minuta settle, larval chambers are enlarged by syncytial tissues containing yolk inclusions. Lipid inclusions at the basal attachment site gradually became smaller during the six weeks of our study. In O. minuta, the flagellated chambers that differentiate in the larva become the post‐metamorphic flagellated chambers, which corroborate the view that internalization of these chambers during embryogenesis is a process that resembles gastrulation processes in other animals.     

Cytopathogenicity of Naegleria fowled for Cultured Rat Neuroblastoma Cells1

The cytopathogenicity of Naegleria fowleri strain LEE (ATCC-30894) for cultured rat neuroblastoma cells (B-103) has been investigated. Both live N. fowleri amoebae and Naegleria lysates added to ⁵¹Cr-labeled B-103 cells caused release of radiolabel, which was dependent upon the ratio of amoebae to target cells or to the lysate concentration. Lysates of N. fowleri strains LEE, NF-66, NF-69, and HB-4 were equally injurious to B-103 target cells whereas lysates of strains 6088 and KUL were less cytotoxic. Highly pathogenic mouse-passaged strain LEE were less cytotoxic than axenically grown amoebae. Maximum cytotoxicity was observed in lysates from amoebae in late exponential or early stationary phase of growth. Cytopathogenicity of lysates was reduced after heating at 44°C for 60 min or at 60°C for 30 min. Cytotoxicity was stable during storage at 4°C or at ?20°C for 26 h. Neither live amoebae nor lysates injured B-103 target cells at 4°C. Live amoebae and lysates injured B-103 by a time, temperature, and concentration dependent process.     

Taxonomic Criteria for Limax Amoebae,with Descriptions of 3 New Species of Hartmannella and 3 of Vahlkampfia*

SYNOPSIS. Seven species of limax amoebae were isolated into clonal, monoxenic cultures with Aerobacter aerogenes from material collected from freshwater habitats. Studies were made of their trophic structure, nuclear division, cyst structure, some aspects of cytochemistry, and other characteristics. Six new species are described: Vahlkampfia inornata, V. avara, V. jugosa, Hartmannella limacoides, H. vermiformis, and H. exundans. The well-known species Naegleria gruberi (Schardinger, 1899) is re-described on the basis of 8 strains; its flagellated phase was found to be biflagellate, with rare exceptions. A correlation exists between the manner of locomotion and the pattern of nuclear division in the limax amoebae in the family Vahlkampfiidae and those in the genus Hartmannella. Trophic amoebae of all species had a PAS-positive surface layer, altho results with H. vermiformis and H. exundans were less definite than with other species. All species except H. limacoides formed cysts in culture. The cyst walls of all cyst-forming species were strongly PAS-positive, but results of the zinc chloroiodide test for cellulose were negative with the method used. The genus Hartmannella Alexeieff, 1912, is re-defined to include those species which assume a simple, monopodial limax-like form during locomotion and have nuclear division similar to that of metazoan cells and to distinguish it from the genus Acanthamoeba Volkonsky, 1931.     

The timing of phenotypic suppression of an aggregation defect by an aggregation-stimulating factor from Polyspboadylium violaceum

Abstract. A class of aggregation deficient mutants of Polysphondylium violaceum ( aggA ) had previously been shown to aggregate in the presence of an excreted, dialyzable product (D factor) prepared from wild type amoebae. We have characterized further the development of the aggA mutants in liquid culture. In the absence of an external source of D factor, aggA mutants never become aggregation competent. D factor must be added to the mutants in order for them to be able to aggregate when removed from liquid culture and plated on a surface. The ability of D factor to stimulate the development of aggregation competence can be illustrated with both the aggA mutant and wild type amoebae. D factor is only required by the aggA mutants at a late stage in development of aggregation competence and does not have to be present continuously during incubation. Wild type amoebae provided with additional D factor become aggregation competent earlier than amoebae incubated without additional D factor. These data suggest that the amoebae develop most of the biochemical functions necessary in order to aggregate and that D factor is necessary to trigger aggregation. One of these biochemical functions, development of aggregation-specific adhesion sites, has been shown to occur in the aggA mutant in the absence of D factor.     

Resumption of locomotion byAmoeba proteus readhering to different substrata

Summary Floating heterotactic cells ofAmoeba proteus were sedimented on untreated glass surfaces and on modified substrata, differing in their wettability and surface potential. About 95% of the amoebae readhere to the glass within 12 min and recover locomotive (polytactic) morphology within 13 min. The rate of locomotion resumption does not change significantly on styrene/methyl methacrylate co-polymers with contrasting hydrophilic sulfonic group surface densities. Almost all amoebae readhere within 3 min to the positively charged surface of polylysine-coated glass, but locomotive shape is only reassumed after 20 min by 95% of them. The polytactic cells are marked flattened on polylysine and move 2 1/2 times more slowly than on the glass. Floating amoebae never readhere to negatively charged gelatin gel; up to 25% become polytactic after 20 min, but they never resume locomotion. Indifference of amoebae to substratum wettability, and their prompt reaction to the positively or negatively charged surfaces, are discussed. The polylysine and gelatin gel substrata seem suitable for the study of adhesion dependent motor functions in amoebae.     

The regulation of glycoprotein synthesis by cAMP in Dictyostelium

Dictyostelium discoideum (Dd) 1-H vegetative amoebae exposed to cAMP differentiate into mature stalk cells within 48 h [6]. It was of interest to monitor the patterns of glycoprotein synthesis in the amoebae during the first 5 h of exposure to cAMP and phosphate buffer (PB) controls. Following the exposure period the amoebae were labeled with -[6-³H]fucose. It was determined by both silver grain counts of autoradiographs and scintillation spectroscopy that within minutes cAMP effects an inhibition of [³H]fucose incorporation. However, by 5 h of exposure both experimentals and controls lose a major amount of their labeling capacity based upon the initial PB control value. Vegetative amoebae exposed to cAMP mimics the sparse labeling found in prestalk cells. Prestalk cells synthesize cellulose as a result of cAMP-induced gluconeogenesis and consequently glycoprotein synthesis is reduced. Cellular interactions promoted by cAMP appears to initiate prestalk cell differentiation during the pre-aggregation phase of development. This event is accompanied by a loss in the ability of the aggregating cells to synthesize glycoprotein.     

The importance of morphological versus chemical defences for the bloom-forming cyanobacterium Microcystis against amoebae grazing

Amoebae grazing can be an important loss factor for blooms of the common cyanobacterium Microcystis. Some Microcystis strains seem to be protected against amoebae grazing, but it is unclear whether this is achieved by their colony morphology or biochemically. These factors were investigated in grazing experiments using two Microcystis-grazing amoebae (Korotnevella sp. and Vannella sp.) and two Microcystis strains with differing colony morphology (aeruginosa and viridis morphotype) and different sensitivity to amoebae grazing. Amoebae did not increase in density and failed to reduce the growth rate of cultures of the amoebae insensitive viridis strain, irrespective of whether the Microcystis strain was colonial or unicellular. This suggests that the extended mucilage matrix surrounding viridis colonies is not the main defence mechanism against amoebae grazing. At the same time, the growth rate of both unicellular and colonial cultures of the amoebae-sensitive aeruginosa strain was heavily reduced by the growing amoebae. The addition of filtered viridis-conditioned medium to aeruginosa cultures significantly decreased both amoebae growth and its effect on aeruginosa growth rates, which indicates that extracellular compounds constitutively produced by viridis are at least partially responsible for their insensitivity to amoebae grazing. These results demonstrate the potential importance of chemical interactions between lower trophic levels (protists) for Microcystis bloom dynamics.     

Chemotactic response of wild-type and aggregation-defective mutants of Polysphondylium violaceum

Abstract. The aggregation-specific chemoattractant for Polysphondylium violaceum is N-propionyl-γ-L-glutamyl-L-ornithine-δ-lactam ethyl ester, or glorin. Wild-type amoebae allowed to develop in liquid culture acquire increased ability to respond to glorin shortly after starvation, i.e., just prior to the time they become aggregation competent. Similarly, as development proceeds, the amoebae show decreased sensitivity to folic acid, but they show almost no response to cyclic AMP at any time during their development in liquid culture. The optimum concentrations for the chemotactic response are 10^-8 M for glorin and 10^-5–10^-6 M for folic acid. A class of aggregation-defective mutants, aggA , will not aggregate in the absence of an excreted pheromone, D factor. During development in liquid culture in the presence or absence of D factor, these aggA mutants show a chemotactic response similar to that of wild-type amoebae to folic acid and glorin. However, D factor does enhance the chemotactic response of aggA mutants to glorin. In the absence of D factor, mutant amoebae will form fruiting bodies if exposed to a chemotactic gradient of either folic acid or glorin. Under these conditions, the mutant amoebae circumvent the requirement for D factor in order to develop.     

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.     

The amoebae of Idionectes vortex (Cutosea,Amoebozoa): Motility,cytoskeleton architecture and extracellular scales

The Cutosea represent a deep-branching lineage within the phylum Amoebozoa that is still relatively poorly explored. Currently, there are four cutosean representatives known – the monotypic genera Armaparvus, Idionectes, Sapocribrum, and Squamamoeba – with marked genetic distances. Idionectes vortex is the deepest-branching species and differs markedly from the other Cutosea in ecology, life history, and most importantly, in its ability to form a flagellated swarmer with an exceptional swimming mechanism. As far as we know, the other Cutosea lack flagella and rather represent small, marine amoebae with a characteristic cell coat. The present paper focuses on the amoeboid life history stage of the algivorous amoeboflagellate Idionectes vortex to provide data for a first in-depth comparison with other Cutosea and to document structural specialties. The amoeboid stage of Idionectes is mainly associated with the specific feeding process, that is, the interaction with algal prey cells and phagocytosis of protoplast material. Yet, the present data from time-lapse microscopy, cytochemical stainings, and electron microscopy demonstrate clear similarities with the other cutosean species concerning amoeboid locomotion and cell coat ultrastructure. Furthermore, Idionectes amoebae exhibit a well-developed microtubular cytoskeleton, and an unusual basal apparatus that seems to undergo marked changes during the life history of this exceptional amoebozoan.     

Fine‐scale spatial ecology drives kin selection relatedness among cooperating amoebae

Cooperation among microbes is important for traits as diverse as antibiotic resistance, pathogen virulence, and sporulation. The evolutionary stability of cooperation against “cheater” mutants depends critically on the extent to which microbes interact with genetically similar individuals. The causes of this genetic social structure in natural microbial systems, however, are unknown. Here, we show that social structure among cooperative Dictyostelium amoebae is driven by the population ecology of colonization, growth, and dispersal acting at spatial scales as small as fruiting bodies themselves. Despite the fact that amoebae disperse while grazing, all it takes to create substantial genetic clonality within multicellular fruiting bodies is a few millimeters distance between the cells colonizing a feeding site. Even adjacent fruiting bodies can consist of different genotypes. Soil populations of amoebae are sparse and patchily distributed at millimeter scales. The fine‐scale spatial structure of cells and genotypes can thus account for the otherwise unexplained high genetic uniformity of spores in fruiting bodies from natural substrates. These results show how a full understanding of microbial cooperation requires understanding ecology and social structure at the small spatial scales microbes themselves experience.