Phylogenetic position of the pelobiont Mastigamoeba aspera and revision of the family Mastigamoebidae (Archamoebae, Pelobiontida).

In this study, we presented the results of our molecular phylogenetic analysis of Archamoebae using a newly obtained sequence of the 18S rRNA gene of Mastigamoeba aspera, the type species of the genus. In addition, we characterized the general organization of the tubulin cytoskeleton of M. aspera by immunofluorescent microscopy and TEM. Our findings allowed us to revise the family Mastigamoebidae and establish within it two subfamilies: Mastigamoebinae for the genus Mastigamoeba and Seraviniinae subfam. nov. for the genera Seravinia gen. nov., Paramastigamoeba gen. nov., Iodamoeba and Endolimax. The type genera Mastigamoeba and Seravinia are clearly distinguished by the structure of the basal apparatus of flagella. In addition, the tubulin cytoskeleton of several Mastigamoeba spp. contains a rim of microtubules around the nuclei.

Glomerulosomes: morphologically distinct nuclear organelles of unknown nature.

The nucleus of some representatives of the genus Pelomyxa (Amoebozoa, Archamoebae, Pelobiontida) contains specific bodies (membrane-less organelles). They may be either embedded in the nucleolar mass or detached from the nucleolus. We termed these nuclear bodies the glomerulosomes for their characteristic ultrastructural appearance. The glomerulosomes are distinct nuclear bodies, about 1 µm in diameter. The morphological and diagnostic unit of a glomerulosome is an electron-dense thread/string, about 30-40 nm in thickness. These threads are not direct continuation of the nucleolar material. The threads create the unique geometric appearance of the glomerulosome by being organized into precisely parallel rows/cords. Each cord of the threads can curve at different angles within the glomerulosome body, but the threads themselves are not coiled. Nowadays, the glomerulosomes have been discovered in P. palustris, P. stagnalis, P. paradoxa, and Pelomyxa sp. Despite the unique appearance of glomerulosomes, their existence may be a more common phenomenon in eukaryotic cells than just a specific feature of the nucleus of elected pelomyxes.

Pelomyxa doughnuta sp. nov. (Archamoebea, Pelobiontida) with an unusual nucleus-glycogen association.

We described Pelomyxa doughnuta sp. nov. and examined it with the use of light, electron, and immunofluorescence microscopy as well as cytochemical methods. The cells of P. doughnuta sp. nov. are usually binuclear, although cells with one, three, or four nuclei are sometimes found in the population. A unique feature of the new species is a dense capsule around the nucleus. It consists of a continuous layer of glycogen 5-20 µm thick. The tubulin cytoskeleton is mainly represented by perinuclear microtubules. P. doughnuta sp. nov. has a filamentous glycocalyx and strongly reduced components of flagellar apparatus. Obligate prokaryotic endocytobionts of two morphotypes are present in the cytoplasm.

Cell-cell fusions and cell-in-cell phenomena in healthy cells and cancer: Lessons from protists and invertebrates.

Herein we analyze two special routes of the multinucleated cells' formation - the fusion of mononuclear cells and the formation of cell-in-cell structures - in the healthy tissues and in tumorigenesis. There are many theories of tumorigenesis based on the phenomenon of emergence of the hybrid cancer cells. We consider the phenomena, which are rarely mentioned in those theories: namely, cellularization of syncytium or coenocytes, and the reversible or irreversible somatogamy. The latter includes the short-term and the long-term vegetative (somatic) cells' fusions in the life cycles of unicellular organisms. The somatogamy and multinuclearity have repeatedly and independently emerged in various groups of unicellular eukaryotes. These phenomena are among dominant survival and biodiversity sustaining strategies in protists and we admit that they can likely play an analogous role in cancer cells.

Associative Conditioning Is a Robust Systemic Behavior in Unicellular Organisms: An Interspecies Comparison.

The capacity to learn new efficient systemic behavior is a fundamental issue of contemporary biology. We have recently observed, in a preliminary analysis, the emergence of conditioned behavior in some individual amoebae cells. In these experiments, cells were able to acquire new migratory patterns and remember them for long periods of their cellular cycle, forgetting them later on. Here, following a similar conceptual framework of Pavlov's experiments, we have exhaustively studied the migration trajectories of more than 2000 individual cells belonging to three different species: Amoeba proteus, Metamoeba leningradensis, and Amoeba borokensis. Fundamentally, we have analyzed several relevant properties of conditioned cells, such as the intensity of the responses, the directionality persistence, the total distance traveled, the directionality ratio, the average speed, and the persistence times. We have observed that cells belonging to these three species can modify the systemic response to a specific stimulus by associative conditioning. Our main analysis shows that such new behavior is very robust and presents a similar structure of migration patterns in the three species, which was characterized by the presence of conditioning for long periods, remarkable straightness in their trajectories and strong directional persistence. Our experimental and quantitative results, compared with other studies on complex cellular responses in bacteria, protozoa, fungus-like organisms and metazoans that we discus here, allow us to conclude that cellular associative conditioning might be a widespread characteristic of unicellular organisms. This new systemic behavior could be essential to understand some key principles involved in increasing the cellular adaptive fitness to microenvironments.

The nature and features of organization of reserve polysaccharides in three Pelomyxa species (Archamoebea, Pelobiontida).

The nature and features of organization of reserve polysaccharides in three species of the genus Pelomyxa-P. palustris, P. belevskii, and P. stagnalis-were studied using light and transmission electron microscopy. We applied the periodic acid-Schiff reaction that is a highly selective method for detecting glycogen. The fluorescent dye auramine-SO2 (Au-SO2) was used as a Schiff-type reagent. The densely packed aggregates of glycogen that form the morphologically differentiated organelle-like bodies are revealed in the cytoplasm in all studied species. The organization of these bodies is characterized by the species-specific features, while in most cases, their size and number in the cells vary depending on the season of the year. Although in all the cases we studied, these bodies do not have their own boundary membrane, in fact, they are surrounded by membranous structures. These structures differ in a variety of Pelomyxa species. We concluded that there are two groups of species in the genus Pelomyxa. The first one includes organisms containing glycogen structures in the cytoplasm (P. palustris, P. belevskii, P. stagnalis, P. binucleata, P. corona, P. secunda). No inclusions resembling glycogen bodies were found in P. flava, P. paradoxa, P. gruberi, and P. prima that form the second group.

Karyotypic instability of endoprophase and mitotic cells of Amoeba sp. strain Cont from the "proteus-type" group (Amoebozoa, Euamoebida, Amoebidae).

We performed karyotyping of Amoeba sp. strain Cont. Based on the results of a cytological analysis, we concluded that the chromosome number of Amoeba sp. strain Cont in mitosis was unstable. In all cases they appeared to be hypergaploid (the basic chromosome number is 30), with monosomy of all chromosomes except four shortest ones. The presence of "extrachromosomes" in the nucleus could prolong until the beginning of the anaphase. It was only then that they were ejected from the nucleus and the euploidy (haploidy) was restored. The stage of endoprophase nucleus was revealed in the cell cycle of Amoeba sp. strain Cont. This stage has not yet been found in other amoebae from the "proteus-type" group that had been previously studied (A. proteus strain B and A. borokensis). The maximum number of endoreplication rounds in the strain Cont amoebae nuclear cycle was 4 or 5. The regular extrusion of chromosomes from the nucleus into the cytoplasm occurred in each of the endoreplication rounds. Comparative cytological analysis of A. proteus strain B, A. borokensis and Amoeba sp. strain Cont karyotypes indicated that strain Cont, though rather close to the former two amoebae, is actually a distinct species.

The Chromatin Extrusion Phenomenon in Amoeba proteus Cell Cycle.

Amoeba proteus is possibly the best known of all unicellular eukaryotes. At the same time, several quintessential issues of its biology, including some aspects of the cell cycle, remain unsolved. Here, we show that this obligate agamic amoebae and related species have a special type of cyclic polyploidy. Their nucleus has an euploid status only for a small fraction of the cell cycle, during metaphase and telophase. The rest of the time it has an aneuploid status, which is a consequence of polyploidization. Extrusion of "excess" chromatin from the nucleus in late interphase and during prophase results in depolyploidization. Such a strategy of life cycle in unicellular eukaryotes is thought to be the main mechanism of "resetting" the Muller's ratchet and is a satisfactory alternative to the meiotic recombination for agamic protists.

Experimental Listeria-Tetrahymena-Amoeba food chain functioning depends on bacterial virulence traits.

BACKGROUND: Some pathogenic bacteria have been developing as a part of terrestrial and aquatic microbial ecosystems. Bacteria are consumed by bacteriovorous protists which are readily consumed by larger organisms. Being natural predators, protozoa are also an instrument for selection of virulence traits in bacteria. Moreover, protozoa serve as a "Trojan horse" that deliver pathogens to the human body. Here, we suggested that carnivorous amoebas feeding on smaller bacteriovorous protists might serve as "Troy" themselves when pathogens are delivered to them with their preys. A dual role might be suggested for protozoa in the development of traits required for bacterial passage along the food chain. RESULTS: A model food chain was developed. Pathogenic bacteria L. monocytogenes or related saprophytic bacteria L. innocua constituted the base of the food chain, bacteriovorous ciliate Tetrahymena pyriformis was an intermediate consumer, and carnivorous amoeba Amoeba proteus was a consumer of the highest order. The population of A. proteus demonstrated variations in behaviour depending on whether saprophytic or virulent Listeria was used to feed the intermediate consumer, T. pyriformis. Feeding of A. proteus with T. pyriformis that grazed on saprophytic bacteria caused prevalence of pseudopodia-possessing hungry amoebas. Statistically significant prevalence of amoebas with spherical morphology typical for fed amoebas was observed when pathogenic L. monocytogenes were included in the food chain. Moreover, consumption of tetrahymenas fed with saprophytic L. innocua improved growth of A. proteus population while L. monocytogenes-filled tetrahymenas provided negative effect. Both pathogenic and saprophytic bacteria were delivered to A. proteus alive but only L. monocytogenes multiplied within amoebas. Observed differences in A. proteus population behaviour suggested that virulent L. monocytogenes might slow down restoration of A. proteus ability to hunt again and thus restrict the size of A. proteus population. Comparison of isogenic bacterial pairs that did or did not produce the haemolysin listeriolysin O (LLO) suggested a role for LLO in passing L. monocytogenes along the food chain. CONCLUSIONS: Our results support the idea of protozoa as a means of pathogen delivery to consumers of a higher order and demonstrated a dual role of protozoa as both a "Trojan horse" and "Troy."

Nucleus-associated actin in Amoeba proteus.

The presence, spatial distribution and forms of intranuclear and nucleus-associated cytoplasmic actin were studied in Amoeba proteus with immunocytochemical approaches. Labeling with different anti-actin antibodies and staining with TRITC-phalloidin and fluorescent deoxyribonuclease I were used. We showed that actin is abundant within the nucleus as well as in the cytoplasm of A. proteus cells. According to DNase I experiments, the predominant form of intranuclear actin is G-actin which is associated with chromatin strands. Besides, unpolymerized actin was shown to participate in organization of a prominent actin layer adjacent to the outer surface of nuclear envelope. No significant amount of F-actin was found in the nucleus. At the same time, the amoeba nucleus is enclosed in a basket-like structure formed by circumnuclear actin filaments and bundles connected with global cytoplasmic actin cytoskeleton. A supposed architectural function of actin filaments was studied by treatment with actin-depolymerizing agent latrunculin A. It disassembled the circumnuclear actin system, but did not affect the intranuclear chromatin structure. The results obtained for amoeba cells support the modern concept that actin is involved in fundamental nuclear processes that have evolved in the cells of multicellular organisms.

Dermamoeba algensis n. sp. (Amoebozoa, Dermamoebidae): an algivorous lobose amoeba with complex cell coat and unusual feeding mode.

The genus Dermamoeba unifies oblong, flattened amoebae of lingulate morphotype, possessing a thick multilayered cell coat. It includes two species, D. granifera and D. minor. In this paper we describe a third species of this genus, D. algensis n. sp. This species is algivorous; engulfing a large algal cell, it destroys part of the cell coat liberating the plasma membrane, which forms the food vacuole. Thus the glycocalyx never appears inside the phagosome. This observation confirms that some of the thick-coated amoebae may use this way to avoid energetically costly digestion of their own glycocalyx. Studies of the physiology of this organism show that it feeds most actively at a temperature of 22-25 °C. Below and above this temperature the feeding intensity drastically decreases. The new species can survive NaCl concentrations up to 5%, which roughly corresponds to 50 ppt salinity. Accordingly, D. algensis has a wide range of salinity tolerance.