Locomotion of Physarum polycephalum amoebae is guided by a short range interaction with E. coli

Studies of the behavior of Physarum polycephalum amoebae have shown that locomotion of these cells is guided by surfaces composed of aggregated bacteria. Amoebae move readily on both E. coli and agar surfaces. However, when a cell migrating on bacteria encounters an edge of the bacterial surface, the orientation of cell movement changes so that the cell maintains contact with bacteria. Time-lapse cinemicrographic studies employing wild type and mutant cells show that this behavior involves short range interactions between amoebae and bacteria, that it is not dependent on variations in the rate of phagocytosis, and that it is not a simple result of constraints on cell movement imposed by adhesive bonds between amoebae and bacteria. These results provide evidence that guidance of cell locomotion depends on active regulation of the cellular force generating system as the amoebae contact surfaces of varying characteristics and, therefore, suggest that this system is amenable to detailed studies of process involved both in cell-cell recognition and in linking such recognition to regulation of cell movement.     

Adhesion to the substratum improves the motility ofAmoeba proteus in the absence of a cell nucleus

Summary Anucleated fragments ofAmoeba proteus obtained by dissection and kept on an untreated glass surface fail to adhere to this substratum, lose motor polarity, and stop moving, at least for several hours. If they are transferred after the operation to a highly adhesive surface (polylysine-coated glass), they adhere to the substratum, although locomotion is not spontaneously restored. However, after exposure to a light-shade difference along their body they start moving towards the shaded area and continue locomotion as long as the photic stimulus is acting. Disorganisation of the F-actin cytoskeleton of anucleated fragments was observed on the untreated glass but reorganization on the polylysine-coated surface. The anucleated fragments can show transient recovery of slight spontaneous motor activity and react promptly to external stimuli after up to several days on untreated glass. These intermittent activity periods are enabled by reconstruction of F-actin cytoskeleton in the anucleated fragments during their temporary adhesion to the glass. It is concluded that the injurious effect of cell nucleus removal on the locomotor capacity of amoebae can be compensated by the simultaneous enhancement of cell adhesion and application of a stimulus restoring the motor polarity of the cell. The compensation is achieved by cytoskeletal reorganization.     

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.     

Amoeboid locomotion of Acanthamoeba castellanii with special reference to cell-substratum interactions

The amoeboid locomotion of Acanthamoeba castellanii has been studied by observation of individual cells moving on a planar glass substratum. Cell-substratum interactions involved in traction have been observed by reflexion interference microscopy. A variable part of the ventral surface of A. castellanii formed a protean platform, the 'associated contact', from which filopodia were subtended; these established stable, focal adhesions (approximately 0.4 micron diameter) on the substratum beneath. Surprisingly, acanthopodia, a prominent feature of this protozoon, did not play an obvious role in traction. The dimensions of the cell-substratum gap in the associated contact could be modulated by the concentration of ambient electrolyte. Dilution of electrolyte from 50 mM-KC1 to 2mM resulted in (i) an increase in the cell-substratum gap, (ii) a marked decrease in cell motility, (iii) reduced cell adhesion to glass.     

Locomotion and phenotypic transformation of the amoeboflagellate Naegleria gruberi at the water-air interface

The protozoon Naegleria gruberi is able to carry out amoeboid locomotion at the water-air interface in a manner indistinguishable from that exhibited on solid substrata with the production of focal contacts and associated filopodia. The speed of locomotion at this interface can be modulated by changes in electrolyte concentrations; these speed changes are identical to those observed at a water-glass interface. The nature of the water-air interface is discussed leading to the hypothesis that surface tension alone could provide suitable properties for the adhesion and translocation of amoebae at this interface without necessitating specific, absorbed molecules. The temporary swimming flagellate stage of Naegleria is able to dock at the interface, make stable adhesions to it, and revert to the amoeboid phenotype. Conversely, amoebae resident at the water-air interface can transform to swimming flagellates and escape into the bulk liquid phase. We report the presence of Naegleria amoebae in the surface microlayers of natural ponds; thus, in freshwater bodies there may be active shuttling of Naegleria amoebae from the benthos to the surface microlayers by means of the non-feeding, swimming flagellate phenotype. The public health implication of this behaviour in the case of the pathogenic relative, Naegleria fowleri, is discussed.     

Cell surface interaction of the protozoan Gregarina with concanavalin A beads - implications for models of gregarine gliding

Con A Sepharose beads can be translocated over the surface of the protozoan Gregarina and in (forward) moving gregarines, the bead may be moved backwards relative to the substrate. The speed of bead movement is not constant over the surface of the cell, but has a maximum value in the central region of the deutomerite. The mass of the individual beads used in this study was about the same order of magnitude as the mass of a gregarine, i.e. considerable force must be generated at the gregarine cell surface. The implications of these experiments to models of gregarine locomotion are considered. The close similarities between this system and flagellar surface motility of Clamydomonas studied by particle movement and gliding motility are discussed.     

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.     

Rho/Rho-dependent kinase affects locomotion and actin–myosin II activity of Amoeba proteus

Summary. The highly motile free-living unicellular organism Amoeba proteus has been widely used as a model to study cell motility. However, the molecular mechanisms underlying its unique locomotion are still scarcely known. Recently, we have shown that blocking the amoebaes endogenous Rac- and Rho-like proteins led to distinct and irreversible changes in the appearance of these large migrating cells as well as to a significant inhibition of their locomotion. In order to elucidate the mechanism of the Rho pathway, we tested the effects of blocking the endogenous Rho-dependent kinase (ROCK) by anti-ROCK antibodies and Y-27632, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride, a specific inhibitor of ROCK, on migrating amoebae and the effect of the Rho and ROCK inhibition on the actin-activated Mg-ATPase of the cytosolic fraction of the amoebae. Amoebae microinjected with anti-ROCK inhibitors remained contracted and strongly attached to the glass surface and exhibited an atypical locomotion. Despite protruding many pseudopodia that were advancing in various directions, the amoebae could not effectively move. Immunofluorescence studies showed that ROCK-like protein was dispersed throughout the cytoplasm and was also found in the regions of actin–myosin II interaction during both isotonic and isometric contraction. The Mg-ATPase activity was about two- to threefold enhanced, indicating that blocking the Rho/Rho-dependent kinase activated myosin. It is possible then that in contrast to the vertebrate cells, the inactivation of Rho/Rho-dependent kinase in amoebae leads to the activation of myosin II and to the observed hypercontracted cells which cannot exert effective locomotion.Correspondence and reprints: Department of Muscle Biochemistry, Nencki Institute of Experimental Biology, 3 Pasteur ulica, 02-093 Warsaw, Poland.     

Discrete movements of foot epithelium during adhesive locomotion of a land snail

During the adhesive locomotion of land snails a series of short dark transverse bands, called pedal or foot waves, is visible ifa moving snail's ventral surface is observed through a sheet of glass. Moreover, the mucus secreted from the pedal glands and some pedal epithelial cells forms a thin layer which acts as a glue augmenting adherence, while also acting as a lubricant under the moving parts of the snail's foot. The relationships between velocity and the frequency of pedal waves as well as changes in the volume of small air bubbles under foot waves were analyzed by means of digital recordings made through a glass sheet on which the snails were moving. On the ventral surface of a moving snail foot, the adhering parts of the foot constituted about 80% of the total area, while several moving parts only about 20%. The single moving region of the foot (the pedal wave) amounted to about 3% of snail length. The epithelium in the region of the pedal wave was arched above the substrate and was also more wrinkled than the stationary epithelium, which enabled the forward motion of each specific point of epithelium during the passage of a pedal wave above it. The actual area of epithelium engaged by a pedal wave was at least 30% greater than the area of the epithelium as recorded through a glass sheet. In the region of the pedal wave, the tiny subepithelial muscles acting on the epithelium move it up in the front part of the wave, and then down at the end of the wave, operating vertically in relation to the substrate. In the middle part of the wave, the epithelium only moves forward. In summary, during the adhesive locomotion of snails, the horizontal movement of the ventral surface epithelium proceeds as temporally separate phases of upward, forward and downward movement.     

Movement of a non-flagellate spermatozoon: A study of the male gamete of Nematospiroides dubius (Nematoda)

The spermatozoa of Nematospiroides dubius were studied using the scanning electron microscope and time-lapse cinematography. Spermatozoa undergo a profound change in morphology after insemination: they change from an elongate structure, 16–18 μm long, to a more rounded form about 5–10 μm in diameter. Spermatozoa from female worms stuck to, and migrated across a glass surface by the production of pseudopodia, but they adhered more readily to a glass surface coated with egg albumin. The average speed of a sample of six differentiated spermatozoa was 7·3 μm/min. Their locomotion is not considered to be amoeboid but resembles the movement of monopodial neutrophils. A hypothesis for the mechanism of movement is presented, and other possible functions of the pseudopodial region are discussed.     

The role of the cell cycle in differentiation of the cellular slime mould Dictyostelium discoideum

Summary During development and differentiation of the cellular slime mould Dictyostelium discoideum there appears to be a relationship between the cell cycle and cell fate: amoebae halted in G2 phase during early development differentiate into spores whereas stalk cells are formed from amoebae halted in GI phase. It is proposed that this is because a major effect of the cell cycle is to generate heterogeneity in the cell surface properties of the developing amoebae.     

Calcium,cyclic GMP and the control of myosin II during chemotactic signal transduction ofDictyostelium

Evidence is presented for Ca²⁺ and cyclic GMP being involved in signal transduction between the cell surface cyclic AMP receptors and cytoskeletal myosin II involved in chemotactic cell movement. Ca²⁺ is shown to be required for chemotactic aggregation of amoebae. The evidence for uptake and/or eflux of this ion being regulated by the nucleotide cyclic GMP is discussed. The connection between Ca²⁺, cyclic GMP and chemotactic cell movement has been explored using “streamer F” mutants. The primary defect in these mutants is in the structural gene for the cyclic GMP-specific phosphodiesterase which results in the mutants producing an abnormally prolonged peak of accumulation of cyclic GMP in response to stimulation with the chernoattractant cyclic AMP. While events associated with production and relay of cyclic AMP signals are normal, certain events associated with movement are (like the cyclic GMP response) abnormally prolonged in the mutants. These events include Ca²⁺ uptake, myosin II association with the cytoskeleton and inhibition of myosin heavy and light chain phosphorylation. These changes can be correlated with the amoebae becoming elongated and transiently decreasing their locomotive speed after chemotactic stimulation. Other mutants studied in which the accumulation of cyclic GMP in response to cyclic AMP stimulation was absent produced no myosin II responses. Models are described in which cyclic GMP (directly or indirectly via Ca²⁺) regulates accumulation of myosin II on the cytoskeleton by inhibiting phosphorylation of the myosin heavy and light chain kinases.     

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.     

Correlation of adenosine phosphate levels with cellular morphology in Dictyostelium discoideum amoebae

Prior to completion of aggregation and the beginning of multicellular differentiation, the amoebae of Dictyostelium discoideum assume two distinct phases with characteristic changes in cellular movement, shape and adhesiveness. These two phases of amoeboid behaviour have been studied with respect to the quantitative analysis of the intracellular adenosine phosphates, using both enzymatic and chromatographic techniques. A higher intracellular ATP level and energy-charge has been found for the actively moving, non-adhesive amoebae as compared to the flattened, mutually adhesive cells. The importance and possible role of ATP in regulating amoeboid form, movement and cell adhesion is discussed.     

Friction and adhesion in the tarsal and metatarsal scopulae of spiders

Friction and adhesion forces of the ventral surface of tarsi and metatarsi were measured in the bird spider Aphonopelma seemanni (Theraphosidae) and the hunting spider Cupiennius salei (Ctenidae). Adhesion measurements revealed no detectable attractive forces when the ventral surfaces of the leg segments were loaded and unloaded against the flat smooth glass surface. Strong friction anisotropy was observed: friction was considerably higher during sliding in the distal direction. Such anisotropy is explained by an anisotropic arrangement of microtrichia on setae: only the setal surface facing in the distal direction of the leg is covered by the microtrichia with spatula-like tips. When the leg is pushed, the spatula-shaped tips of microtrichia contact the substrate, whereas, when the leg is pulled over a surface, setae bend in the opposite direction and contact the substrate with their spatulae-lacking sides. In an additional series of experiments, it was shown that desiccation has an effect on the friction force. Presumably, drying of the legs results in reduction of the flexibility of the setae, microtrichia, spatulae, and underlying cuticle; this diminishes the ability to establish proper contact with the substrate and thus reduces the contact forces.     

Pathogenic and Nonpathogenic Acanthamoeba spp. in Thermally Polluted Discharges and Surface Waters1

During spring and autumn, the total number of amoebae and the number of Acanthamoeba species able to grow at 37°C were determined in six thermally polluted factory discharges and the surrounding surface waters. The isolated Acanthamoeba strains were studied for growth in axenic medium, cytopathic effect in Vero cell cultures, and virulence in mice. Although more amoebae were isolated in autumn, the number of Acanthamoeba species was lower than in spring, when the percent of pathogenic strains among the isolates was highest. Higher concentrations of amoebae were found in warm discharges, and more virulent strains occurred in thermal discharges than in surface waters.     

Pseudoparamoeba garorimi n. sp., with Notes on Species Distinctions within the Genus

A new marine species of naked lobose amoebae Pseudoparamoeba garorimi n. sp. (Amoebozoa, Dactylopodida) isolated from intertidal marine sediments of Garorim Bay, Korea was studied with light and transmission electron microscopy. This species has a typical set of morphological characters for a genus including the shape of the locomotive form, type of subpseudopodia and the tendency to form the single long waving pseudopodium in locomotion. Furthermore, it has the same cell surface structures as were described for the type species, Pseudoparamoeba pagei: blister‐like glycostyles with hexagonal base and dome‐shaped apex; besides, cell surface bears hair‐like outgrowths. The new species described here lacks clear morphological distinctions from the two other Pseudoparamoeba species, but has considerable differences in the 18S rDNA and COX1 gene sequences. Phylogenetic analysis based on 18S rDNA placed P. garorimi n. sp. at the base of the Pseudoparamoeba clade with high PP/BS support. The level of COX1 sequence divergence was 22% between P. garorimi n. sp. and P. pagei and 25% between P. garorimi n. sp. and P. microlepis. Pseudoparamoeba species are hardly distinguishable by morphology alone, but display clear differences in 18S rDNA and COX1 gene sequences.     

Relative motion inAmoeba proteus in respect to the adhesion sites. I. Behavior of monotactic forms and the mechanism of fountain phenomenon

Summary The unbranched ectoplasmic cylinder of monotacticA. proteus is always retracted toward the cell-substrate attachment sites. The retraction velocity increases from the adhesion sites toward any free distal body end in a linear way, which indicates the uniform contractility of the whole cylinder. Therefore, in the cells frontally attached all the ectoplasm moves forward, and in those adhering by the tail the whole ectoplasmic tube moves backward producing the full fountain phenomenon. With cell attachment at the middle body regions, which is most typical for normal locomotion, the whole ectoplasm is centripetally retracted from both body poles toward the adhesion zone, producing then the tail retraction in the posterior and incomplete fountain in the anterior body part. In unattached amoebae the whole peripheral tube is retracted toward its geometrical centre which coincides with its posterior closed end, producing therefore also a full fountain. It is generalized that the fountain arises always between an unattached front and the nearest attachment point behind its manifestation zone. The photographic records of movement and longitudinal velocity profiles of ectoplasmic retraction are identical on both sides of the attachment points, suggesting the same mechanism for the fountain movement as for the tail withdrawal. It is concluded therefore that not the axial endoplasmic arm of the fountain is active, but its peripheral arm built of the ectoplasm.All elements complicating the cell contour, as the constriction rings and ephemeral lateral pseudopodia, do not change their position in respect to the ectoplasmic material, but move together with it in respect to the substrate, i.e., the cytoskeleton moves as a whole. Loose glass rods attached by adhesion to cell surface also precisely follow the cytoskeleton movements, being transported toward the main locomotory adhesion zone established on the firm substrate, although the cell membrane as such behaves differently. It suggests a direct connection between the adhesion sites and the cytoskeleton.Study supported by Research Project II. 1 of the Polish Academy of Science.I dedicate this paper to the memory of Reginald J. Goldacre, deceased in December 1983, who twenty years ago introduced me to the study of amoebae.     

A laboratory study of the correlation between texture and the speed of locomotion by the sea urchin Hemicentrotus pulcherrimus

This laboratory study examined the relationship between substrate texture and movement speed of the sea urchin Hemicentrotus pulcherrimus. We assessed the movement speed of 14 sea urchins placed on either acrylic, or three types of waterproof sandpaper, on the bottom of a water tank. Images were taken at regular intervals and analyzed to determine the speed of each sea urchin. Light intensity was stronger at one end of the tank. Our analysis showed sea urchins moved away from light, at a speed that was negatively correlated with the roughness of the substrate, with slower movement on rougher surfaces. This result has implications for the design of equipment for capturing sea urchins in areas where their explosive population growth presents a threat to algal growth and reef environments.