Extraction of an actomyosin-like pootein from amoebae of Dictyostelium discoideum

An actomyosin-like protein has been extracted from amoebae of Dictyostelium discoideum, V-12. The purified protein exhibited a reversible change in viscosity upon addition of ATP, indicating an ATP sensitivity of 75–85% and a specific viscosity of 0.1. At low ionic strength in the presence of Mg⁺⁺ and ATP the amoeba protein displayed the phenomenon of superprecipitation. The protein extract was found to be an adenosinetriphosphatase (ATP'ase) hydrolyzing ATP to ADP and inorganic phosphate. Both Mg⁺⁺ and Ca⁺⁺ at low ionic strength accelerated the ATP ase activity whereas at high ionic strength only Ca⁺⁺ stimulated ATP hydrolysis. The ATP'ase activity was inhibited by ethylene-diamine-tetraacetic-acid, Mersayl and p-chloromercuribenzoate. The physico-chemical and enzymatic properties of the extracted amoeba protein are qualitatively comparable to those of muscle actomyosin, and very similar in quantitative properties to smooth muscle actomyosin and the actomyosin-like proteins of blood platelets, leucocytes and slime mold plasmodia. The significance of the presence of this actomyosin-like protein in Dictyostelium amoebae is discussed in relation to amoeboid form and movement.     

Speed of locomotion of the soil amoebaNaegleria gruberi in media of different ionic compositions with special reference to interactions with the substratum

Summary The effect of electrolytes on the speed of locomotion of amoebae on glass coverslips was studied in a perfusion chamber. The intervening gap between the ventral surface of the cell and the glass substrate in the various solutions was also studied by means of reflexion interference microscopy. It was found that the presence of electrolyte increased the speed of movement,e.g., the amoebae moved almost four times faster in 10 mM KCl than in deionized water. The ventral surface of the amoebae was brought closer to the substrate when increasing amounts of electrolyte were present in the medium. This two fold effect was fully reversible by replacement of electrolyte with deionized water. Solutions of the non-electrolytes sucrose or urea did not affect the behaviour of the amoebae with respect to their speed of movement or cell-substrate separation distance compared with that observed in deionized water. Thus we have demonstrated a direct relationship between the speed of amoeboid cell locomotion and the closeness of the cell to the substrate.     

Diverse Legionella‐Like Bacteria Associated with Testate Amoebae of the Genus Arcella (Arcellinida: Amoebozoa)

Diverse species of Legionella and Legionella‐like amoebal pathogens (LLAPs) have been identified as intracellular bacteria in many amoeboid protists. There are, however, other amoeboid groups such as testate amoeba for which we know little about their potential to host such bacteria. In this study, we assessed the occurrence and diversity of Legionella spp. in cultures and environmental isolates of freshwater arcellinid testate amoebae species, Arcella hemispherica, Arcella intermedia, and Arcella vulgaris, via 16S rRNA gene sequence analyses and fluorescent in situ hybridization (FISH). Analysis of the 16S rRNA gene sequences indicated that A. hemispherica, A. intermedia, and A. vulgaris host Legionella‐like bacteria with 94–98% identity to other Legionella spp. based on NCBI BLAST search. Phylogenetic analysis placed Legionella‐like Arcella‐associated bacteria (LLAB) in three different clusters within a tree containing all other members of Legionella and LLAPs. The intracellular localization of the Legionella within Arcella hosts was confirmed using FISH with a Legionella‐specific probe. This study demonstrates that the host range of Legionella and Legionella‐like bacteria in the Amoebozoa extends beyond members of “naked” amoebae species, with members of the testate amoebae potentially serving an ecological role in the dispersal, protection, and replication of Legionella spp. in natural environments.     

The life cycle of the amoeboid alga Synchroma grande (Synchromophyceae, Heterokontophyta)--highly adapted yet equally equipped for rapid diversification in benthic habitats

Synchroma grande (Synchromophyceae, Heterokontophyta) is a marine amoeboid alga, which was isolated from a benthic habitat. This species has sessile cell stages (amoeboid cells with lorica and cysts) and non‐sessile cell stages (migrating and floating amoebae) during its life cycle. The different cell types and their transitions within the life cycle are described, as are their putative functions. Cell proliferation was observed only in cells attached to the substrate but not in free‐floating or migrating cells. We also characterised the phagotrophy of the meroplasmodium in comparison to other amoeboid algae and the formation of the lorica. The functional adaptations of S. grande during its life cycle were compared to the cell stages of other amoeboid algae of the red and green chloroplast lineages. S. grande was found to be highly adapted to the benthic habitat. One sexual and two asexual reproductive strategies (haplo‐diploid life cycle) support the ability of this species to achieve rapid diversification and high adaptivity in its natural habitat.     

Lipid requirements for axenic cultivation ofEntamoeba histolytica

Fatty acids, cholesterol and glucose present in axenic medium are utilized by growingEntamoeba histolytica but the amoeba is unable to synthesize cholesterol from [U^-14 C^- ] glucose although the label is incorporated into the fatty acids and non-saponifiable fractions of the organism. Exogenously-added sonicated dispersions of cholesterol, Β-sitosterol, lanosterol, lecithin and lauric, palmitic, linoleic and stearic acids are ingested by the amoebae with subsequent loss in amoeboid movement. After a few hours the movement is regained. Cholesterol, lecithin and the fatty acids stimulate amoebic multiplication but are unable to replace serum in the medium either singly or in combination. CDRI Communication No. 2516.     

The activation of cell aggregation by phosphorylation in Dictyostelium discoideum

Single amoebae of D. discoideum are phosphorylated in the presence of external ATP. Phosphorylation is catalyzed by a cAMP independent cell membrane bound protein kinase. As a result of phosphorylation cell aggregation is induced and the chemotactic sensitivity of the amoebae to a cAMP gradient decreased. Cell membrane phosphorylation may be involved in triggering cell aggregation in vivo. The fact that the number of free phosphorylable sites per cell decreases at the onset of aggregation gives support to this hypothesis. The existence of a plasma membrane bound phosphoprotein phosphatase suggests a possible regulator role for this enzyme on the phosphorylation of the amoebae. Finally, ATP inhibits intercellular contact sites outside the aggregation center. Despite this inhibiting effect on cell adhesiveness, amoebal movement toward an aggregation center maintains its normal periodicity.     

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.     

The Life Cycle of Cryptochlora perforans (Chlorarachniophyta)

Observations on the behaviour of different life cycle stages, gamete fusions, and measurements of nuclear DNA contents in Cryptochlora perforans resulted in a first concept concerning life histories in Chlorarachniophyta: the life cycle of Cr. perforans is diplohaplontic (gamete fusion with karyogamy - mitosis - meiosis - mitosis). In the haploid as well as in the diploid life cycle phases amoeboid and coccoid stages occur. The isomorphic gametes are modified amoebae frequently without filopodia. Only haploid flagellate stages are known representing mito- or meiozoospores. Diploid coccoid stages have a granular cytoplasmic structure and may be somewhat larger than haploid ones. Nevertheless, positive identification of haploid (gametophytic) and diploid (sporophytic) stages is only possible on the basis of nuclear DNA contents.     

The fine structure of a species of the amoebo-flagellate Pseudospora Cienk

Summary Mixed cultures of the amoebo-flagellate and the alga on which it feeds were examined by electron microscopy. Both amoeboid and flagellate stags were sectioned and their morphology described, particular attention being paid to the flagellar bases and to the intra-nuclear fibrils. The latter are discussed with relation to nuclear fibrils in other organisms. The amoeba is compared with other amoebae whose fine structure has been examined, and the possible phylogeny of Pseudospora is considered.     

Locomotion and feeding of Acanthamoeba at the water-air interface of ponds

Acanthamoeba trophozoites attach to and effect amoeboid locomotion at the water-air interface of ponds. Their locomotory rate (approximately 0.8 microm s(-1)) and manner of independent movement at this interface is similar to that over solid substrata. Adhesion forces developed between amoebae and the water-air interface are greater than gravity and thus amoebae are also transported passively without detachment. Amoebae docked with the water-air interface remain and flourish here as they are shown, by using green fluorescent protein-labelled Aeromonas hydrophila, to feed on bacteria that occur at the interface, digesting them intracellularly.     

Patterns in the distribution of intracellular ATP concentration in relation to coordination of amoeboid cell behavior in Physarum polycephalum

The Physarum plasmodium reacts tactically to external stimuli. The cell behavior of this giant amoeboid cell was studied by analysing intracellular ATP concentration. The two-dimensional (2D) spatial distribution of ATP depended on cell shape: a polar pattern for a unidirectionally migrating plasmodium, a bowl shape for a circular plasmodium, a hump shape for an oval plasmodium, or a wavy pattern for plasmodia stimulated with blue light or confined in a small chamber, etc. Local external stimulation brought about new patterns of ATP distribution. The ATP concentrations around the stimulated frontal region were reduced by about a half stimulation with KCl (repellent) or casamino acids (attractant). In both cases, migration was inhibited. Migration velocity increased almost linearly with increasing concentration of intracellular ATP above the threshold (about 20 micrograms/mg protein). Under anaerobic conditions or at low temperatures, the intracellular ATP oscillated slowly with a periodicity of about 30 min. Pattern formations in the intracellular ATP concentration and amoeboid coordination are discussed in terms of coupled chemical oscillators in a self-organizing system.     

Obligate amoebae of the protostelids: significance for the concept of Eumycetozoa

Most flagellate Eumycetozoa have a non-flagellate, or obligately amoeboid, trophic state which differentiates from the amoebo-flagellate state and gives rise to the fruiting body. This study examines the morphology, general ultrastructure, and microtubular systems of the obligate amoebae of three flagellate protostelids with typical amoebo-flagellate states. The obligate amoebae of all three species are morphologically distinct indicating that the obligately amoeboid state has evolved independently in the history of each species. Therefore, obligate amoebae may be useful for defining separate evolutionary groups within the Eumycetozoa.     

Rapid redistribution of myosin II in livingDictyostelium amoebae,as revealed by fluorescent probes introduced by electroporation

Summary Fluorescently labeled myosin II fromDictyostelium and fluorescently labeled antibody Fab fragments against myosin II fromDictyostellium were introduced into livingDictyostelium amoebae by electroporation. Fluorescent labeling of myosin II impairs neither actin-activated ATPase activity nor the ability to form filaments in vitro. Fluorescently labeled Fab also did not interfere with the functions of myosin II in vitro. After electroporation, introduced fluorescently labeled myosin II was distributed diffusely in the endoplasm but some of it accumulated at the tail cortical region of migrating cells. During the course of observations, intense fluorescence due to myosin II disappeared and then it appeared again instantaneously in the cortical regions during amoeboid movement. Fluorescently labeled Fab, after electroporation, bound to endogenous myosin II in amoebae and the dynamic changes in its distribution were similar to those of fluorescently labeled myosin II. The fluorescence due to myosin II also underwent dynamic redistribution during the division of cells and chemotactic stimulation. The introduction of labeled Fab and labeled myosin II did not impair the motility ofDictyostelium. During changes in direction associated with cell locomotion, myosin II accumulated at the original front region of the cell and, thereafter, the accumulation was observed at the new tail region of the cell. These results are consistent with the hypothesis that myosin II has two possible roles for cell locomotion. One is that myosin II accumulates at tail regions to produce the power required for contraction. The other is that it hinders the extension of pseudopods in directions other than the frontal direction.     

Variation in the SSUrDNA of the Genus Protostelium Leads to a New Phylogenetic Understanding of the Genus and of the Species Concept for Protostelium mycophaga (Protosteliida,Amoebozoa)

Members of the genus Protostelium (including P. mycophaga, P. nocturnum, and P. okumukumu) are protosteloid amoebae commonly found in terrestrial habitats on dead plant matter. They, along with the closely allied nominal genus Planoprotostelium, containing the single species Pl. aurantium, all have an amoeboid trophic stage with acutely pointed subpseudopodia and orange lipid droplets in the granuloplasm. These amoebae form stalked fruiting bodies topped with a single, usually deciduous spore. The species are identified based on their fruiting body morphologies except for Pl. aurantium which looks similar to P. mycophaga in fruiting morphology, but has amoebae that can make flagella in liquid medium. We built phylogenetic trees using nuclear small subunit ribosomal DNA sequences of 35 isolates from the genera Protostelium and Planoprotostelium and found that (1) the nonflagellated P. nocturnum and P. okumukumu branch basally in the genus Protostelium, (2) the flagellate, Pl. aurantium falls within the genus Protostelium in a monophyletic clade with the nominal variety, P. mycophaga var. crassipes, (3) the cultures initially identified as Protostelium mycophaga can be divided into at least three morphologically recognizable taxa, P. aurantium n. comb., P. apiculatum n. sp., and P. m. rodmani n. subsp., as well as a paraphyletic assemblage that includes the remainder of the P. mycophaga morphotype. These findings have implications for understanding the ecology, evolution, and diversity of these amoeboid organisms and for using these amoebae as models for other amoeboid groups.     

Pseudopodium activation and inhibition signals in chemotaxis by Dictyostelium discoideum amoebae

The behaviour of Dictyostelium discoideum amoebae has been studied in natural cAMP waves and in controlled spatial and temporal gradients. Chemoattractant gradients induce responses which indicate that amoebae spatially compare concentration increases at different points on the cell surface. This allows them to respond to the relative spatial and temporal gradients in a manner that is little affected by the absolute attractant concentration over several orders of magnitude. The changes in turning behaviour, motility and morphology that are induced by attractant gradients are consistent with transduction of stimuli into two intracellular signals - one activating and the other inhibiting pseudopodium formation. The former measures the present attractant concentration at particular points on the cell surface - the local, current signal. The latter measures the average attractant concentration over the whole cell surface during the recent past - the global, past signal. Both signals may be part of a normal pseudopodium autoactivation and inhibition system responsible for amoeboid morphology and motility. Attractants could modulate this system to generate the complex behavioural responses observed.     

ATP and the autonomy of the contractile vacuole in Amoeba proteus

Contractile vacuole function in amoebae treated with immobilizing (5 mM) and nonimmobilizing (0.125 mM) concentrations of ATP has been studied. In ATP-immobilized amoebae, most vacuolar parameters are accelerated, especially the rate of output which passes from 30 to 70 micron3/sec. This favors the concept of an autonomous vacuole, fully functional in the absence of any bulk contribution to it from remote points of the cell. A lower concentration of ATP (0.125 mM), which does not inhibit movement, causes a still greater acceleration of vacuolar function. Work is in progress to elucidate the site and mode of action of exogenous ATP on Amoeba.     

Oscillations in cell shape and size during locomotion and in contractile activities of Physarum polycephalum, Dictyostelium discoideum, Amoeba proteus and macrophages

Changes in cell shape and size were measured during locomotion, together with the motive force of the protoplasmic streaming, in various amoeboid cells in different stages of their life cycle, and under various environmental conditions. The variations in these measurements with time were examined by Fourier spectral analysis. Notwithstanding a change in cell type in the life cycle of P. polycephalum, myxamoebae and tiny plasmodia showed a similar time pattern of locomotion, exhibiting oscillations having a mixture of several periods. A regular oscillation with protoplasmic streaming appeared in the plasmodium only above a critical cell size. D. discoideum amoebae oscillated with two periods of a few minutes in preaggregation stage, but with a period of 10 min in aggregation stage, the latter being induced by cAMP. Macrophages and A. proteus also oscillated with periods of a few minutes. Periods of all these oscillations were prolonged severalfold by respiratory inhibition with NaCN, but were unaffected by glycolytic inhibition with 2-deoxyglucose. Cell fragments of A. proteus containing fewer granules oscillated more slowly and with a larger amplitude than those containing more granules. Among the granules, the nucleus was excluded as a possible modifier of the oscillation. The oscillation in Physarum plasmodium was reversibly suppressed by combining respiratory and ATPase inhibitions in mitochondria with NaCN and oligomycin, intracellular ATP concentration being kept at an appropriate level. The present results show that amoeboid motility, as well as cell shape, is oscillatory and that mitochondria are involved in time keeping.     

THE LIFE CYCLE AND TOXICITY OF PFIESTERIA PISCICIDA REVISITED1

Despite use of excellent molecular techniques, Litaker et al. (2002) cannot provide insights about the life history of toxic Pfiesteria piscicida because they showed no data in support of having used toxic strains; rather they presented evidence that they used non‐inducible strains. Litaker et al. did not find amoeboid stages or a chrysophyte‐like cyst stage in several cultures and unequivocally concluded that the stages do not exist in all P. piscicida strains. Thus, they did not consider the tenet that absence of evidence does not constitute proof of absence. Apparent discrepancies between the research by Litaker et al. and previous research on Pfiesteria can be resolved as follows: First, Litaker et al. did not use toxic strains. We have reported findings (similar to Litaker et al.) showing few amoeboid transformations in non‐inducible strains, which manifest some but not all of the forms that have been documented in some toxic strains. We, and others, have documented active toxicity to fish, transformations to amoebae, and chrysophyte‐like cysts in some clonal toxic strains. Second, the data from several recent publications, which were available but not mentioned by Litaker et al. or by Coats (2002) in accompanying commentary, have verified P. piscicida amoebae, chrysophyte‐like cysts, and other stages in some toxic strains through a combination of approaches including PCR data from clonal cultures.     

Molecular Evolutionary Analyses of Nuclear-Encoded Small Subunit Ribosomal RNA Identify an Independent Rhizopod Lineage Containing the Euglyphina and the Chlorarachniophyta

The Rhizopoda comprise a diverse assemblage of protists which depend on lobose or filose pseudopodia for locomotion. The biochemical and morphological diversity of rhizopods has led to an uncertain taxonomy. Ribosomal RNA sequence comparisons offer a measure of evolutionary relatedness that is independent of morphology and has been used to demonstrate a polyphyletic origin of the Lobosea. We sequenced complete small subunit ribosomal RNA coding regions from the filose amoebae, Euglypha rotunda and Paulinella chromatophora (Euglyphina) to position these taxa in the eukaryote phylogeny. The neighbor-joining analyses show that E. rotunda and P. chromatophora share a monophyletic origin and are not closely related to any lobose amoebae in our analyses. Instead, the Euglyphina form a robust sister group to the Chlorarachniophyta. These results provide further evidence for the polyphyly of the Rhizopoda and support the creation of a new amoeboid lineage which includes the Euglyphina and the chlorarachniophyte algae; taxa with tubular mitochondrial cristae and filose or reticulate pseudopodia.