Visualization of actin polymerization and depolymerization cycles during polyamine-induced cytokinesis in living Amoeba proteus

Summary Microinjection of spermine induces cytokinesis of Amoeba proteus. Within 30–60 s after spermine injection cells form one, or less commonly, two cleavage furrows and within the following 4–10 min the constrictions are completed. The resulting nucleated cell parts show normal streaming and locomotion, whereas the non-nucleated cell parts remain stationary and later degenerate.The intracellular distribution of fully polymerization-competent fluorescently labelled muscle actin was followed by image intensification. Double injection experiments initially using labelled actin and 30 min later spermine revealed a ring-like structure of enhanced fluorescence corresponding to the constricting cleavage furrow. Immediately after cleavage was completed, the ring disappeared. Electron microscopy of cells fixed during spermine-induced cytokinesis showed numerous well aligned actin and myosin filaments in the developing cleavage furrow. These filaments are a specialized manifestation of the cell cortex.The results demonstrate that cycles of actin and myosin polymerization and depolymerization and the parallel alignment of preexisting filaments (crosslinking) represent a basic mechanism in the generation of the motive force during cytokinesis.     

Spatial organization and fine structure of the cortical filament layer in normal locomoting Amoeba proteus

Summary The fine structural organization of a cortical filament layer in normal locomoting Amoeba proteus was demonstrated using improved fixation and embedding techniques. Best results were obtained after application of PIPES-buffered glutaraldehyde in connection with substances known to prevent the depolymerization of F-actin, followed by careful dehydration and freeze-substitution.The filament layer is continuous along the entire surface; it exhibits a varying thickness depending on the cell polarity, measuring several nm in advancing regions and 0.5–1 m in retracting ones. Two different types of filaments are responsible for the construction of the layer: randomly distributed thin (actin) filaments forming an unordered meshwork beneath the plasma membrane, and thick (myosin) filaments mostly restricted to the uroid region in close association with F-actin.The cortical filament layer generates the motive force for amoeboid movement by contraction at posterior cell regions and induces a pressure flow that continues between the uroid with a high hydrostatic pressure and advancing pseudopodia with a low one. The local destabilization of the cell surface as a precondition for the formation of pseudopodia is enabled by the detachment of the cortical filament layer from the plasma membrane. This results in morphological changes by the active separation of peripheral hyaloplasmic and central granuloplasmic regions.     

Dynamics of the cytoskeleton inAmoeba proteus: II. Influence of different agents on the spatial organization of microinjected fluorescein-labeled actin

Summary Iodoacetamido-fluorescein-(IAF)-labeled actin was microinjected into normal locomotingAmoeba proteus. Thereafter (30–60 minutes) changes in the cytoplasmic fluorescence distribution pattern and contractile activity were induced by internal and external chemical stimulation. Different agents such as phalloidin, procaine, 2.4-dinitrophenol (DNP), puromycin, ouabain and n-ethyl maleimide (NEM) interfere with the excitation-contraction mechanism involved in ordered pseudopodium formation during ameboid movement and cause various morphogenetic reactions based on actin polymerization-depolymerization cycles. Most frequent changes are (a) local condensation of IAF-actin and formation of a continuous IAF-actin layer at the cytoplasmic surface of the cell membrane and around the pulsating vacuole, (b) immobilization and hyalo-granuloplasm separation by combined contraction and detachment of the IAF-actin layer from the cell membrane, (c) organized and disorganized formation of pseudopodia by local contraction and disintegration of the IAF-actin layer, and (d) alterations in the rheological properties of the protoplasmic matrix by changes in the molecular state of soluble actin not incorporated into the cytoskeleton. The experimental approaches to the function of the actomyosin system in large amebas attainable by the method ofin vivo molecular cytochemistry are discussed in detail with respect to the participation of the cytoskeleton in motive force generation for cytoplasmic streaming and ameboid movement.     

Effects of the actin-binding protein DNAase I on cytoplasmic streaming and ultrastructure of Amoeba proteus

Microinjection of DNAase I, which is known to form a specific complex with G-actin, induces characteristic changes in cytoplasmic streaming, locomotion and morphology of the contractile apparatus of A. proteus. Light microscopical studies show pronounced streaming originating from the uroid and/or the retracting pseudopods, which ceases 10--15 min after injection of DNAase I, at a time when ultrasctructural studies show that the actin filament system is very much reduced. These results suggest that a controlled reversible equilibrium between soluble and polymerized forms of actin is a necessary requirement for amoeboid movement. The topographic distribution of contractile filaments beneath the plasma membrane visualized by correlated light- and electron microscopy of DNAase I-injected cells establishes the importance of the membrane-bound filamentous layer for three major aspects of streaming: (1) Streaming originates by local contractions of a cell membrane-associated filament layer at the uroid and/or retracting pseudopods, creating a pressure flow. (2) This flow continues beneath the membrane, which is stabilized by filaments in the lateral regions between the posterior end, with a high hydrostatic pressure, and the anterior end, with a low hydrostatic pressure. (3) Pseudopods or extending areas are created by a local destabilization of the cell periphery caused by the separation of the filamentous layer from the plasma membrane.     

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.     

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.     

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     

Binding characteristics of receptors for phagocytosis on the surface of Amoeba proteus

Summary Binding of the tripeptide n-formylmethionyl-leucylphenylalanine (NFMLP) to phagocytic receptors on the surface of Amoeba proteus was examined. Peptide-binding is reversible and demonstrates saturation kinetics. The receptors for phagocytosis are internalized by a temperature-sensitive process with indications that the receptors are recycled. The amoeba is capable of down-regulating its receptors for phagocytosis in response to constant external peptide levels, and also increasing the number of surface receptors in response to food deprivation. On the basis of competition studies, there is evidence that Amoeba proteus has separate surface receptors for both pinocytosis and phagocytosis.     

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.     

Actin dynamics in Amoeba proteus motility

Summary. We studied the distribution of the endogenous Arp2/3 complex in Amoeba proteus and visualised the ratio of filamentous (F-actin) to total actin in living cells. The presented results show that in the highly motile Amoeba proteus, Arp2/3 complex-dependent actin polymerisation is involved in the formation of the branching network of the contractile layer, adhesive structures, and perinuclear cytoskeleton. The aggregation of the Arp2/3 complex in the cortical network, with the exception of the uroid and advancing fronts, and the spatial orientation of microfilaments at the leading edge suggest that actin polymerisation in this area is not sufficient to provide the driving force for membrane displacement. The examined proteins were enriched in the pinocytotic pseudopodia and the perinuclear cytoskeleton in pinocytotic amoebae. In migrating amoebae, the course of changes in F-actin concentration corresponded with the distribution of tension in the cell cortex. The maximum level of F-actin in migrating amoebae was observed in the middle-posterior region and in the front of retracting pseudopodia. Arp2/3 complex-dependent actin polymerisation did not seem to influence F-actin concentration. The strongly condensed state of the microfilament system could be attributed to strong isometric contraction of the cortical layer accompanied by its retraction from distal cell regions. Isotonic contraction was limited to the uroid. Correspondence and reprints: Department of Molecular and Cellular Neurobiology, Nencki Institute of Experimental Biology, Polish Academy of Sciences, ulica Pasteura 3, 02-093 Warszawa, Poland.     

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.     

The ecological energetics of Amoeba proteus (Protozoa)

The consumption, production and respiration of Amoeba proteus were measured in the laboratory for cells cultured over a range of Tetrahymena pyriformis concentrations (125–4 000 cells/0.5 ml) at 10, 15 and 20 °C. Differences were attributed to both temperature and prey availability. A series of generation energy budgets were constructed for amoebae grown under the above conditions. The biological efficiencies linking the parameters of the budget equation were calculated. Assimilation efficiencies ranged from 22–59% regardless of temperature. Net production efficiencies were high at 15 and 20 °C (65–82%) but low at 10 °C (11–49%). Gross production efficiencies were also higher at 15 and 20 °C (16–47%) than at 10 °C (4–29%). The ecological implications of this investigation are discussed.     

Diacylglycerol as a component of the signal-coupling pathway during the initiation of endocytosis in Amoeba proteus

In an attempt to define the transmembrane-signal pathway used to couple external phagocytotic signals with effectors in the cell interior, the effects of diacylglycerol (DG) and related substances were examined in Amoeba proteus. DGs are highly chemotactic, readily attracting amoebae when presented in a glass micropipette. Addition of DG (10^-6 M) to the medium elicits rapid shape changes in the amoeba and the formation of large phagosomes. Monacylglycerol and 1,3-diacylglycerol were much less effective in eliciting phagosome formation. On the assumption that DG was stimulating phosphokinase C (PKC) activity in the amoeba, the effect of phorbol myristate acetate (PMA), a known activator of PKC activity i other cell systems, was assessed in the amoeba. PMA (10^-7 M) alone was capable of bringing about shape changes in amoebae as well as stimulating the formation of phagosomes. These observations suggest that PKC is involved in the signal-coupling associated with the onset of phagocytosis. On the other hand staurosporine and H-7, inhibitors of PKC activity in some cell systems, did not inhibit the phagocytic uptake of Tetrahymena by A. proteus. It may be then that DGs in the amoeba interact directly with elements of the cytoskeleton causing phagosome formation, although a role for PKC in the initiation of phagocytosis in the amoeba cannot be ruled out at this point.     

Morphological evidence for the existence of a more complex cytoskeleton in Amoeba proteus

Summary Various stabilization and extraction procedures were tested to demonstrate the ultrastructural organization of the cytoskeleton in normal, locomoting Amoeba proteus. Most reliable results were obtained after careful fixation in glutaraldehyde/lysine followed by prolonged extraction in a polyethylene glycol/Triton X-100 solution. Before dehydration in a graded series of ethanol and critical-point drying, the amoebae were split by the sandwich-technique, i.e., by mechanical cleavage of cells mounted between two poly-L-lysine-coated glass slides. Platinum-carbon replicas as well as thin sections prepared from such cell fragments revealed a cytoskeleton composed of at least four different types of filaments: (1) 5–7-nm filaments organized as a more or less ordered cortical network at the internal face of the plasma membrane and probably representing F-actin; (2) 10–12-nm filaments running separately or slightly aggregated through the cytoplasm and probably representing intermediate filaments; (3) 24–26-nm filaments forming a loose network and probably representing microtubules; and (4) 2–4-nm filaments as connecting elements between the other cytoskeleton constituents. Whereas microfilaments are responsible for protoplasmic streaming and other motile phenomena, the function of intermediate filaments and cytoplasmic microtubules in amoebae is still obscure.     

Effects of induced pinocytotic activity and extreme temperatures on the morphology of golgi bodies in Amoeba proteus

Summary The morphology of the Golgi apparatus of Amoeba proteus can be influenced by substances inducing pinocytotic activity as well as by extreme temperatures. During the ingestion of a solution of 0.5% egg white the number of Golgi bodies decreases from 100% measured in control cells to 82% measured in cells showing induced pinocytosis. Simultaneously the ratio of the surface area of the cisternae at the proximal face to that of the vesicles at the distal face of single dictyosomes remains constant (1.74–1.72).The decrease and increase of the temperature of the culture medium to 4° C and 30° C respectively, causes the disappearance of most of the dictyosomes. After keeping the cells for 3–10 h at these temperatures the number of Golgi bodies was only 5–10% of the controls. A continued treatment with cold or warm culture medium leads to a partial reorganization of dictyosomes. After 15 h the number of Golgi bodies counted per cell returned to 57% at 4° C and 38% at 30° C. The ratio of the surface area of the Golgi cisternae to the surface area of the Golgi vesicles also alters under the influence of extreme temperatures. The values measured after treating the cells for 3 h, 4 h 10 h and 15 h at 4° C and 30° C amounted to 0.75, 0.85, 1.14 1.53 and 0.93, 0.38, 0.88, 1.60, respectively, compared to 1.72 of control amoebae.The different values of the ratio of the surface area of cisternae to that of vesicles indicate that there are strong morphological changes of single dictyosomes.     

Characterisation of the Rac/PAK pathway in Amoeba proteus

Summary. Molecular mechanisms underlying the unique locomotion of the highly motile Amoeba proteus still remain poorly understood. Recently, we have shown that blocking the endogenous amoebal Rac-like protein(s) leads to distinct and irreversible changes in the appearance of these large migrating cells as well as to a significant inhibition of their locomotion. To elucidate the mechanism of the Rac pathway in Amoeba proteus, we tested the effects of blocking the endogenous myosin I heavy chain kinase (MIHCK), one of the Rac effectors in Acanthamoeba castellanii and Dictyostelium discoideum, with anti-MIHCK antibodies in migrating amoebae, as well as the effect of inhibiting Rac and MIHCK on the actin polymerisation process. Antibodies against A. castellanii MIHCK detected an A. proteus protein with a molecular mass (ca. 95 kDa) similar to the A. castellanii kinase. The cellular distribution of MIHCK in A. proteus was very similar to those of Rac-like protein in amoebae and MIHCK in A. castellanii. Amoebae microinjected with anti-MIHCK antibodies moved slower and protruded fewer wide pseudopodia (5–6) than the control cells (9–10), resembling to some extent the phenotype of cells microinjected with anti-Rac antibodies. The in vitro studies indicate that the A. proteus Rac-like protein, but not the MIHCK isoform, is engaged in the regulation of the nucleation step of the actin polymerisation process. These observations suggest that MIHCK may be one of the effectors for Rac in these extremely large cells. Correspondence and reprints: Department of Muscle Biochemistry, Nencki Institute of Experimental Biology, 3 Pasteur ulica, 02-093 Warsaw, Poland.     

Characteristics of motive force derived from trajectory analysis of Amoeba proteus

Summary. We used a monochromatic charge-coupled-device camera to observe the migration behavior of Amoeba proteus every 5 s over a time course of 10000 s in order to investigate the characteristics of its centroid movement (cell velocity) over the long term. Fourier transformation of the time series of the cell velocity revealed that its power spectrum exhibits a Lorentz type profile with a relaxation time of a few hundred seconds. Moreover, some sharp peaks were found in the power spectrum, where the ratios of any two frequencies corresponding to the peaks were expressed as simple rational numbers. Analysis of the trajectory using a Langevin equation showed that the power spectrum reflects characteristics of the cell’s motive force. These results suggest that some phenomena relating to the cell’s motility, such as protoplasmic streaming and the sol–gel transformation of actin filaments, which seem to be independent phenomena and have different relaxation times, interact with each other and cooperatively participate in the generation process of the motive force. Correspondence and reprints: Department of Biological Sciences, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta 4259, Midoriku, Yokohama 226-8501, Japan.     

New aspects of membrane dynamics of Amoeba proteus contractile vacuole revealed by vital staining with FM 4-64

Summary. The contractile vacuole (CV) cycle of Amoeba proteus has been studied by phase contrast and electron microscopy. However, the understanding of membrane dynamics in this cycle is still poor. In this study, we used live imaging by fluorescence microscopy to obtain new insights. We succeeded in staining the CV with a styryl dye, FM 4-64 (N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide), and obtained the following results. (1) The CV membrane was directly stained with the dye in the external medium when the CV pore opened upon contraction. This indicates that transfer of plasma membrane to the CV does not occur. (2) The membrane dynamics during the CV cycle were elucidated. In particular, the fluorescent CV membrane was maintained as an aggregate just after contraction and the vacuole re-formed from the aggregate. Staining was maintained during continued contraction cycles. We conclude that the CV membrane is maintained during the CV cycle. Correspondence and reprints: Department of Life Science, Graduate School of Life Science, University of Hyogo, Harima Science Park City, Hyogo 678-1297, Japan.     

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.     

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.