Velocity distribution of the anterograde and retrograde transport of extracellular particles byAmoeba proteus

Summary The transverse velocity profiles of the anterograde flow of particles on the cell surface and around it are approximately parabolic. The peak velocity is recorded close to the membrane and the descendent arm of the profile is viscosity-dependent. It indicates that the extracellular forward flow is probably generated by a forward movement of the fluid fraction of the membrane itself. The retrograde component of extracellular movements is manifested by particles kept on the cell surface by adhesion, which behave exactly as the ectoplasmic layer on the opposite side of the membrane,i.e., they probably reflect the movement of that fraction of the surface material which is attached to the cortical microfilaments. In the longitudinal profile, the velocity of anterograde flow rises from the tail to the front of amoeba, but is generally related to the effective cell locomotion rate and not to the movements of any intracellular layer. Around the cells deprived of any attachment to the substratum, which cannot locomote but manifest vigorous intracellular movements, the anterograde flow ceases at least along 2/3 of their lenght. It persists, however, around the frontal fountain zone, where other particles still move backwards together with the retracted ectoplasmic layer. This indicates that the role of the forward flow of and on the cell surface is to compensate for: (1) the increase of the surface area in the frontal regions due to locomotion, (2) the withdrawal of a part of material which is hauled back by the retracting cortical layer. A comprehensive scheme of the velocity distribution within the different layers of a moving amoeba and around it has been constructed on the basis of present and earlier data.Study supported by the Research Project CPBP 04.01 of the Polish Academy of Science.I dedicate this paper to Professor K. E. Wohlfarth-Bottermann with the best wishes for his 65th birthday.     

Locomotion ofAmoeba proteus after standardizing its body shape

Summary Amoeba proteus obliged to follow dark stripes in the form of Y may be studied in three repeatable simple configurations: 1. tail + 1 advancing front, 2. tail + 2 advancing pseudopodia, 3. tail +1 advancing pseudopodium + 1 contracting pseudopodium. Formation of two advancing pseudopodia and the later conversion of one of them into a contracting pseudopodium affect the rate of movement of all the other body parts in the manner predictible by the hydrodynamic concept of the endoplasmic flow in amoeba. An active front stops and begins to retreat when arriving to a constant distance from the posterior body end. The locomotion is disfavoured if new pseudopodia deviate from the former body axis at the angle wider than 35°.Study supported by Research Project II. 1 of the Polish Academy of Science.     

Motory effects of perforating peripheral cell layers ofAmoeba proteus

Summary Perforation of peripheral cell layers ofA. proteus in any place provokes immediate endoplasm efflux, what supports the view that the hydrostatic pressure is higher in the cell interior than outside. The local effusion of endoplasm results in the reversal of flow in formerly advancing pseudopodia, in agreement with the pressure gradient theories of protoplasmic streaming. Amoebae with destroyed frontal zones squeeze all their endoplasm out through the breach, what disproves the frontal contraction hypothesis of amoeboid movement, but supports the concept of a general contraction of cell cortex.Study supported by the Research Project II.1 of the Polish Academy of Science.     

Amoeba proteus displays a walking form of locomotion

This report deals with observations on the directional locomotion of amoeba before and after fixation and scanning electron microscopy. The study was aimed at visualization of the stepwise events of directional movements. After the analysis of the data it is proposed that the amoeba undergoes a sequence of movement events that can be defined as a walking form of locomotion.     

Growth temperature acclimation byAmoeba proteus: Effects on cytoplasmic organelle morphology

Summary The effects on subcellular morphology of maintaining amoebae at temperatures other than 20 C (the routine culture temperature) were assessed. Estimations of cycling potential at each temperature confirmed that acclimation had affected gross cell functioning. Generation times ranged from no division at 6 C, to an optimal minimum of 2 days at 22 C.Organelle morphology changes were studied after 5 days of growth at the new temperatures; alterations were most evident at the extremes of 6 and 28 C. The main mitochondrial alteration resulted in changes to the ratio of Type I: Type II organelles; with a decrease in Type I forms away from the optimal range of 20–22 C. Extended culturing at 6 C generated mitochondrial matrical inclusions. Ribosomal attachment to the endoplasmic reticulum, a common feature of 20 C-grown cells, decreased at the temperature extremes, where an increase in free ribosomes occurred. Upon extended culture at 6 C helical structures, usually observed in groups only within the nucleus, were also present in the cytoplasm. Golgi complexes were less common in cells maintained at extreme temperatures and often showed differences in shape. These changes were all reversible on a return to culturing at 20 C.The possible functional significance of these findings is discussed.     

Reversal of motory polarity ofAmoeba proteus by suction

Summary When a glass capillary is introduced into the posterior body region ofA. proteus and its orifice is maintained inside the flowing mass of endoplasm, an applied suction force invariably initiates the reversal of streaming direction. This initial effect depends as well on the negative pressure value as on the terminal diameter of the pipette. Further transformations of configuration of pseudopodia are due to mixed effects of the direct application of sucking force and of the active response of amoeba to the new situation. When the sucking pipettes are applied to the outer cell surface, probably only a fraction of the negative pressure may be transmitted to the cell interior. The portion of cell periphery exposed to negative pressure acting from outside is still capable to contract. As a result, when amoeba as a whole is progressively sucked into the capillary, it manifests a clear active escape behaviour.Study supported by the Research Project II. 1 of the Polish Academy of Science.     

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.     

Chaotic behavior in the locomotion ofamoeba proteus

Summary The locomotion ofAmoeba proteus has been investigated by algorithms evaluating correlation dimension and Lyapunov spectrum developed in the field of nonlinear science. It is presumed by these parameters whether the random behavior of the system is stochastic or deterministic. For the analysis of the nonlinear parameters, n-dimensional time-delayed vectors have been reconstructed from a time series of periphery and area ofA. proteus images captured with a charge-coupled-device camera, which characterize its random motion. The correlation dimension analyzed has shown the random motion ofA. proteus is subjected only to 3–4 macrovariables, though the system is a complex system composed of many degrees of freedom. Furthermore, the analysis of the Lyapunov spectrum has shown its largest exponent takes positive values. These results indicate the random behavior ofA. proteus is chaotic and deterministic motion on an attractor with low dimension. It may be important for the elucidation of the cell locomotion to take account of nonlinear interactions among a small number of dynamics such as the sol-gel transformation, the cytoplasmic streaming, and the relating chemical reaction occurring in the cell.     

Depolarization of the cell membrane causes inhibition of cell locomotion and pinocytosis inAcanthamoeba castellanii andAmoeba proteus

Summary 10 M CCCP protonophore in an acidic medium causes depolarization of the cell membrane and immediate cessation of locomotion inAcanthamoeba castellanii andAmoeba proteus. In the basic media there is no depolarization or inhibition of cell locomotion. Other depolarizing agents (alkali cations, crown molecules) also stop locomotion and induce pinocytosis in amoeba. Pinocytotic uptake of horseradish peroxidase byAcanthamoeba castellanii is increased by 69% in the presence of CCCP in the medium at pH 5.7 but is not influenced at higher pH values. This might indicate that both amoeboid locomotion and pinocytosis are controlled by membrane potential.     

Pinocytosis and locomotion in amoebae XIV. Demonstration of two different receptor sites on the cell surface ofAmoeba proteus

Summary Two different receptor sites, located on the cell surface ofAmoeba proteus were detected by using fluorescent analog cytochemistry (FAC) and electron microscopy (EM). Bovine serum albumin labeled with fluoresceine-isothiocyanate (FITC-BSA) and unlabeled ferritin bind, in a pH-dependent manner, as cations at the outer filaments of the mucous layer. The anionic receptor sites show a high affinity for Ca-ions which suppress the binding capacity of FITC-BSA and ferritin at low pH-values. The cation receptors obviously play an important role in the initiation of pinocytosis as demonstrated by the internalization, intracellular translocation and sequestration of the FITC-BSA. FITC- or ferritin-labeled concanavalin A (FITC-Con A, ferritin-Con A) bind predominantly in a pH-independent manner at the tips of the outer filaments and the basal zone of the mucous layer. The binding capacity of FITC-Con A is not influenced by external Ca-ions. Other lectins such asDolichos bifloris agglutinin (DBA), peanut agglutinin (PNA),Ricinus communis agglutinin I (RCA I), soybean agglutinin (SBA),Ulex europaeus agglutinin I (UEA I) and wheat germ agglutinin (WGA) are not specifically bound to the cell surface. So far, no experimental evidence has been gathered for the definitive function of a Con-A receptor in the mucos layer ofAmoeba proteus.Abbreviations BSA bovine serum albumin - Con A concanavalin A - CTC chlorotetracycline - DBA Dolichos bifloris agglutinin - DTE dithioeritritol - FITC fluorosceine-isothiocyanate - IEP iso electric point - PIPES 1-4-piperazine-diethane sulfonic acid - PNA peanut agglutinin - RCA I Ricinus communis agglutinin I - SBA soybean agglutinin - Uac uranylacetat - UEA I Ulex europaeus agglutinin I - WGA wheat germ agglutinin     

Assessment of overall metabolism inAmoeba proteus measured by a microcalorimetric method

Summary We have carried out calorimetric determinations of the overall metabolism ofAmoeba proteus. There was no significant difference in metabolic activity between cells that were starved 3, 4, and 5 days. After 7 and 10 days a significantly lower metabolism was found (p < 0.05).The mean value of heat production rate (thermal power) for the cells after 3 days starvation was found to be 0.84 ± 0.14 nW/cell. Optimal number of cells in the ampoule (1,500–4,800) was accompanied by a steady-state power-time curve. With higher cell concentrations (> 5,000) the power-time curve showed an initial peak. The fall in heat production after about 1 hour varied between 30–60%. The decrease in power value was much larger than expected from cell mortality during the calorimetric experiment.Increasing number of cells (range 1,500–10,000) in the calorimetric ampoule caused a decrease of heat production rate per cell. The correlation coefficient was r=–0.85(p < 0.001). The coefficient of variation of the method was found to be 4.8 %. It seems that the use of microcalorimetric technique can be valuable in recording metabolic events in protozoes.     

Effects of bivalent cations on the initiation of Na-induced pinocytosis inAmoeba proteus

Summary EGTA in moderate concentrations, sufficient to remove all Ca²⁺ from the cell surface, blocks pinocytosis. But in higher concentrations of EGTA, which chelate also Mg²⁺, the pinocytosis reappears and is strongly enhanced. Simultaneous removal of both Ca and Mg ions by EDTA brings about only potentiating effect. Reintroduction of either Ca or Mg separately, demonstrates that Mg²⁺ is a powerful inhibitor of pinocytosis. The influence of chelators on the pinocytosis is attributed respectively to their selective or unspecific influence on both bivalent ions at the cell surface, without affecting the intracellular contraction mechanism.Study supported by the Research Project II. 1 of the Polish Academy of Science.     

Ultrastructure of mitosis inAmoeba proteus

Summary The fine structure ofAmoeba proteus nuclei has been studied during interphase and mitosis. The interphase nucleus is discoidal, the nuclear envelope is provided with a honeycomb layer on the inside. There are numerous nucleoli at the periphery and many chromatin filaments and nuclear helices in the central part of nucleus.In prophase the nucleus becomes spherical, the numerous chromosomes are condensed, and the number of nucleoli decreases. The mitotic apparatus forms inside the nucleus in form of an acentric spindle. In metaphase the nuclear envelope loses its pore complexes and transforms into a system of rough endoplasmic reticulum cisternae (ERC) which separates the mitotic apparatus from the surrounding cytoplasm; the nucleoli and the honeycomb layer disappear completely. In anaphase the half-spindles become conical, and the system of ERC around the mitotic spindle persists. Electron dense material (possibly microtubule organizing centers—MTOCs) appears at the spindle pole regions during this stage. The spindle includes kinetochore microtubules attached to the chromosomes, and non-kinetochore ones which pierce the anaphase plate. In telophase the spindle disappears, the chromosomes decondense, and the nuclear envelope becomes reconstructed from the ERC. At this stage, nucleoli can already be revealed with the light microscope by silver staining; they are visible in ultrathin sections as numerous electron dense bodies at the periphery of the nucleus.The mitotic chromosomes consist of 10 nm fibers and have threelayered kinetochores. Single nuclear helices still occur at early stages of mitosis in the spindle region.     

Cytotoxicity of polyamines to Amoeba proteus: Role of polyamine oxidase

It has been shown that oxidation of polyamines by polyamine oxidases can produce toxic compounds (H₂O₂, aldehydes, ammonia) and that the polyamine oxidase-polyamine system is implicated, in vitro, in the death of several parasites. Using Amoeba proteus as an in vitro model, we studied the cytotoxicity to these cells of spermine, spermidine, their acetyl derivatives, and their hypothetical precursors. Spermine and N ¹-acetylspermine were more toxic than emetine, an amoebicidal reference drug. Spermine presented a short-term toxicity, but a 48-h contact time was necessary for the high toxicity of spermidine. The uptake by Amoeba cells of the different polyamines tested was demonstrated. On the other hand, a high polyamine oxidase activity was identified in Amoeba proteus crude extract. Spermine (theoretical 100%) and N ¹-acetylspermine (64%) were the best substrates at pH 9.5, while spermidine, its acetyl derivatives, and putrescine were very poorly oxidized by this enzyme (3–20%). Spermine oxidase activity was inhibited by phenylhydrazine (nil) and isoniazid ( 50%). Mepacrine did not inhibit the enzyme activity at pH 8. Neither monoamine nor diamine oxidase activity ( 10%) was found. It must be emphasized that spermine, the best enzyme substrate, is the most toxic polyamine. This finding suggests that knowledge of polyamine oxidase specificity can be used to modulate the cytotoxicity of polyamine derivatives. Amoeba proteus was revealed as a simple model for investigation of the connection between cytotoxicity and enzyme activity.Abbreviations DAO diamine oxidase - DFMO DL--difluoromethylornithine - DP 1-3-diaminopropane - IC₅₀ 50% inhibition concentration - MAO monoamine oxidase - N ¹-ACSP; N ¹-acetylspermine - N¹-ACSPD N ¹-acetylspermidine - N ⁸-ACSPD N ⁸-acetylspermidine - ODC ornithine decarboxylase - PAO(s) polyamine oxidase(s) - PUT putrescine - SP spermine - SPD spermidine     

Movement of surface markers along the pinocytotic pseudopodia ofAmoeba proteus

Summary The movement of latex beads over pinocytotic pseudopodia produced byAmoeba proteus was recorded in the presence of 117.65 mM EGTA as an inducer of pinocytosis. The results show that all particles flow in the direction of pseudopodial growth, with a slightly higher velocity than the advancing frontal edge. This means that markers are removed from the base of a pinocytotic pseudopodium and gradually approach the pseudopodium tip. Two particles on the surface of the same pseudopodium can move at the same rate or differ slightly in the velocity of their forward flow. A bead can move even if another blocks the channel orifice. Retrograde particle movement has never been observed. Whether all latex spheres bound to pinocytotic pseudopodia flow with the laterally mobile plasma membrane fraction, which slides over submembranous contractile layer, or whether the whole cortical complex, the actin network and the plasma membrane, move together towards the invagination site is discussed.     

Calcium movements associated with peptide induced phagocytosis inAmoeba proteus

Summary Calcium controls the level of phagocytosis inAmoeba proteus which is inhibited by substances such as verapamil and flunarazine which interfere with Ca⁺⁺ movements in a variety of cell systems. Calcium ion movements in amoebae under control conditions appear to be primarily diffusive in response to activity and electrical potential gradients. Chemotactic peptides (n-formylmethionylleucylphenylalanine, NFMLP) at high concentrations (10^–5 M) induce phagocytosis in the amoeba and bring about a concomitant increase in⁴⁵Ca influx. Verapamil, flunarazine and La⁺⁺⁺ all block the increased⁴⁵Ca influx caused by NFMLP, as well as inhibiting phagocytosis. It is suggested that initiation of phagocytosis in the amoeba is associated with the movement of Ca⁺⁺ into the cytoplasm from the external medium resulting in a transient increase in the cytoplasmic Ca⁺⁺ ion activity.     

Chemical stimulation of phagocytosis in Amoeba proteus and the influence of external calcium

Summary The process of phagocytosis in Amoeba proteus was examined by following the uptake of Tetrahymena pyriformis and agarose beads. The ciliates are taken up in a time dependent and saturable manner. T. pyriformis apparently emits a water-soluble substance that acts as a chemoattractant to the amoebae. Plain agarose beads are not engulfed by A. proteus, but those beads having reducedglutathione with the -SH group exposed are taken up almost to the same extent as T. pyriformis. Phagocytosis of the glutathione beads is calcium-dependent with maximum bead uptake at 10^-4M Ca⁺⁺. Glutathione applied to A. proteus brings about pseudopod formation, increased phagocytosis and displacement of surface-associated calcium.     

Pinocytosis and locomotion of amoebae: XII. Dynamics and motive force generation during induced pinocytosis in A. proteus

Summary The mechanism of induced pinocytosis was investigated in Amoeba proteus by light and electron microscopy. The application of nine different inducing substances revealed that pinocytotic channel formation, elongation, vesiculation, shortening and disappearance are the result of the successive or simultaneous action of both traction and pressure forces, which are produced by the contractile activity of a plasma membrane-associated layer of filaments ranging from a few hundred nm to several in thickness. The initial phase of channel formation is caused by traction forces according to the membrane flow concept, whereas channel elongation and vesiculation mainly result from pressure forces in conjunction with the extrusion of small hyaline pseudopodia. Shortening and disappearance of the pinocytotic channels are brought about by local contractions of the cortical filament layer in the basal region of the hyaline pseudopodia. Experiments using latex beads as marker particles together with inducing substances show that a rapid membrane turnover during pinocytosis can be excluded, and that the plasma membrane slides as an entire structure over the underlying cytoplasm.The authors are most grateful to Mrs. J. Ruch for technical assistance     

Prostaglandins may play a signal-coupling role during phagocytosis in Amoeba proteus

Summary Phagocytosis in Amoeba proteus can be induced with prostaglandins (PG). In addition, arachidonic acid (the fatty acid precursor to the PG-2 series) also induces phagocytosis. The induction of phagocytosis with arachidonic acid can be partially inhibited by the cyclooxygenase inhibitor indomethacin. Phagocytosis in the amoeba can also be induced with the chemotactic peptide N-formylmethionyl-leucylphenylalanine (NFMLP). The peptide presumably induces phagocytosis by interacting with receptors on the amoeba surface, which may initiate the release of arachidonic acid from membrane lipids. NFMLP-induced phagocytosis can also be partially inhibited by indomethacin. It is suggested that PG's or biochemically related substances may play a signal-coupling role during phagocytosis in the amoeba.