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.     

Relative motion inAmoeba proteus in respect to the adhesion sites

Summary The whole ectoplasmic layer of polytactic and heterotactic forms ofA. proteus behaves as self-contractile structure. Depending on the configuration of cell body and on the cell-to-substrate attachment conditions it continuously retracts from each distal cell projection toward its centre and/or from each free body end toward the actual adhesion sites. As in the monotactic forms, it leads to the withdrawal of the tail region behind the retraction center and may result in the fountain movement in front of it. In the long unattached pseudopodia of heterotactic forms the ectoplasm is retracted in the fountain form, with the velocity linearly increasing from the basis of pseudopodium up to its tip. In polytactic cells the fountain is often absent, if the advancing fronts immediately adhere to the substrate. When they develop in unattached condition, or are experimentally obliged to detach, the ectoplasmic cylinders of frontal pseudopodia are retracted backwards. On the substrates which do not offer firm points of support the cell periphery moves back as a whole,i.e., the principal ectoplasmic cylinder retracts together with the cylinders of lateral pseudopodia, and the direction and speed of movement in any spot is the resultant of forces produced by all other segments. The retraction of ectoplasmic gel layer is independent of the endoplasmic flow in such extent that a pseudopodium may be withdrawn as a whole in spite of the endoplasm streaming directed forwards in its interior. On the cell surface the particles attached by adhesion (glass rods) strictly follow the movements of the internal ectoplasmic structures, whereas the unattached particles flow forward in the direction of endoplasm streaming.Study supported by Research Project II. 1 of the Polish Academy of Science.     

Dynamics of the contractile system in the pseudopodial tips of normally locomoting amoebae,demonstrated in vivo by video-enhancement

Summary Behaviour of the membrane and contractile system was directly recorded in the advancing and retracting frontal zones of spontaneously locomoting or stimulated amoebae. The advancing pseudopodial tips alternately slow down and accelerate. In the slowing phase the frontal hyaline caps are flat and compressed by countercontraction of the cortical actin network beneath the leading edge. At this stage the membrane-cytoskeleton complex splits: the detached contractile layer is retracted inwards, and the membrane lifted outwards. The fluid endoplasm fraction is filtered forward through the detached actin network. This results in a local hydrostatic pressure drop, immediately restores the forward flow of endoplasm and initiates the acceleration phase of the leading edge progression. The frontal membrane, temporarily disconnected from the cytoskeletal layer, is free to slide and extend forward, but the new submembrane contractile network is soon repolymerized. In this way, after making one step forward, the frontal zone recovers its former state, and the cycle is then repeated. The cortex disassembly-reassembly cycles at the leading edge are produced every 2 s, on average. Retraction of the frontal contractile layers is part of the general centripetal cortex flow observed during motor functions of amoebae and many other cells, and is therefore associated with various other backward movements observed within and on the surface of advancing frontal zones of amoebae. The backward movement of the contractile cortex is also responsible for the withdrawal of previously advancing pseudopodia, if the detachment of successive contractile sheets from the frontal membrane ceases. It was demonstrated that the action of attractants and repellents is based on the activation or inhibition, respectively, of rhythmic disassembly of the membrane-cytoskeleton complex at the leading edge.     

L'Organisation Ultrastructurale Corticale de la Gregarine Lecudina pellucida; ses Rapports avec l'Alimentation et la Locomotion

SYNOPSIS. The structure of the cortical region (epicyte and ectoplasm) of the gregarine Lecudina pellucida , an intestinal parasite of the polychete worm Perinereis cultrifera was studied by electron microscopy.
The epicitary folds have 3 unit type membranes. Between the 1st and 2nd is a layer probably composed of fine longitudinal fibrils which has an arch-like or gutter-like structure at the crest of the folds. Inside these folds is cytoplasm without any noticeable differentiation or inclusion except for a granular (or finely fibrillar) layer under the limiting inner membrane and close to it.
The ectoplasmic zone of the entocyte is separated from the epicitary region by a lengthwise discontinuous cylindrical opaque layer, inwardly tangential to the folds. The ectoplasm lacks paraglycogen granules but has various organelles: apparently pinocytic vesicles against the wall between the folds, vesicles with myelinic membranes, opaque granules, a few mitochondria with blistered internal vesicles, and a few circular tubular fibers.
The superficial zone of the gregarine is supposed to contribute to nutrition, thru the extensive surface furnished by its folds and thru the pinocytic vesicles; but this alimentary intake is incomplete compared with that of the previously studied anterior region.
Insufficient mucus is discharged to account for locomotion. There are some circular ectoplasmic fibers, but locomotory myonemes are completely absent. However, there are deformations of the folds and corresponding waves that could account for locomotion by creeping or swimming. These movements of the folds might be due to the action of the contractile proteins and correspond with some of the layers seen in the wall.     

Ultrastructure and Description of a Fungus-Feeding Amoeba,Trichamoeba mycophaga n. sp. (Amoebidae,Amoebea), from Australia1

ABSTRACT. An amoeba isolated from a wheatfield and a forest soil in Australia has been identified as Trichamoeba mycophaga n. sp. Trophozoites of this amoeba are palmate to elongate and measure 45–136 μm in length and 25–94 μm in width. Amoebae in continuous locomotion may be limax with a villous-bulb uroid. Both the lobose pseudopodia and the advancing margin of a limax trophozoite bear an ectoplasmic crescent. The plasma membrane is coated with an electron-dense amorphous layer ca. 100 nm thick. Endoplasm is granular with elongate to bipyramidal crystals and contains bacterial endosymbionts. Trophozoites have a single, spherical to oval nucleus, 4–10 μm in diameter, which contains a centrally located, spherical to oval nucleolus, 2.8–5.0 μm in diameter. The nucleoplasm contains aggregations of filaments distributed radially within the nuclear membrane. Cysts are 21–60 μm in diameter, with ecto- and endocyst walls separated by an amorphous layer.     

Protein kinase D-mediated anterograde membrane trafficking is required for fibroblast motility

BACKGROUND: Locomoting cells exhibit a constant retrograde flow of plasma membrane (PM) proteins from the leading edge lamellipodium backward, which when coupled to substrate adhesion, may drive forward cell movement. However, the intracellular source of these PM components and whether their continuous retrograde flow is required for cell motility is unknown.RESULTS: To test the hypothesis that the anterograde secretion pathway supplies PM components for retrograde flow that are required for lamellipodial activity and cell motility, we specifically inhibited transport of cargo from the trans-Golgi network (TGN) to the PM in Swiss 3T3 fibroblasts and monitored cell motility using time-lapse microscopy. TGN-to-PM trafficking was inhibited with a dominant-negative, kinase-dead (kd) mutant of protein kinase D1 (PKD) that specifically blocks budding of secretory vesicles from the TGN and does not affect other transport pathways. Inhibition of PKD on the TGN inhibited directed cell motility and retrograde flow of surface markers and filamentous actin, while inhibition of PKD elsewhere in the cell neither blocked anterograde membrane transport nor cell motile functions. Exogenous activation of Rac1 in PKD-kd-expressing cells restored lamellipodial dynamics independent of membrane traffic. However, lamellipodial activity was delocalized from a single leading edge, and directed cell motility was not fully recovered.CONCLUSIONS: These results indicate that PKD-mediated anterograde membrane traffic from the TGN to the PM is required for fibroblast locomotion and localized Rac1-dependent leading edge activity. We suggest that polarized secretion transmits cargo that directs localized signaling for persistent leading edge activity necessary for directional migration.     

Participation of the contractile system in endocytosis demonstrated in vivo by video-enhancement in heat-pretreated amoebae

Summary The heat-pretreated amoebae (hyalospheres) are well suited cell models to study several manifestations of endocytosis: invagination of initial funnels, formation of pinocytotic channels, their activity and disintegration, production of microand macroendosomes directly from the surface membrane. All these phenomena are rhythmically reproduced (with periods ranging from 9 to 27 s) at the same active spots on the cell surface and accompanied by pulsation of the adjacent peripheral cytoplasmic layers. Successive portions of the contractile cortical network are serially detached from the plasma membrane and retracted inwards (on average 1 detachment per 15 s). They are suggested to be responsible for the traction component of endocytotic movements, i.e., for pulling the initial invagination funnels, elongation of channels, and inward transport of macroendosomes which are embedded in them. On the other hand, retraction of the cortical network squeezes the hyaloplasm outwards and thus the pressure component of endocytosic is produced. This results in cell surface expansion around the orifice of endocytotic channels or formation of macroendosomes by constriction at the mouth of large surface invaginations. Moreover, the retracting cortical network produces various radial transhyaline strands which seem to play a, not fully understood, role in membrane invagination and inward transport of microendosomes, and to accompany cytoplasmic pulsation around channels. The contractile network lining the walls of the channels may be detected in vivo, when some old channels are destroyed and their membrane dissociates from the cytoskeletal sleeve. The central role of the rhythmic detachment of the contractile network from the plasma membrane is common to the locomotory and endocytotic movements.     

Entamoeba Motility: Dynamics of Cytoplasmic Streaming, Locomotion and Translocation of Surface-Bound Particles, and Organization of the Actin Cytoskeleton in Entamoeba invadens

ABSTRACT. The dynamics of cytoplasmic streaming, retrograde translocation of externally bound particles and locomotion by Entamoeba invadens were compared. Locomoting amoebae were monopodial, exhibited fountain flow cytoplasmic streaming and translocated externally bound erythrocytes to the rear of cells. The rates of rearward flow of peripheral cytoplasmic vacuoles and of the externally bound particles were equal to the rate of cell forward locomotion. Rhodamine-phalloidin staining revealed a distinct cortical polymerized actin cytoskeleton. This was least evident about the periphery of the advancing pseudopod, increased in density toward the rear of the cell and was most concentrated in the uroid. A monoclonal anti-eucaryotic actin antibody, which recognized monomeric Entamoeba actin on immunoblots, stained trophozoites by indirect immunofluorescence throughout the cytoplasm, but not in the cortical regions stained by rhodamine-phalloidin. This and other evidence implied that the antibody recognized only unpolymerized actin in Entamoeba . We propose that locomotion, cytoplasmic streaming and translocation of externally bound particles are driven by a common actin-based mechanism in Entamoeba , possibly involving retrograde cortical actin flow and recycling.     

Locomotive mechanism of Physarum plasmodia based on spatiotemporal analysis of protoplasmic streaming

We investigate how an amoeba mechanically moves its own center of gravity using the model organism Physarum plasmodium. Time-dependent velocity fields of protoplasmic streaming over the whole plasmodia were measured with a particle image velocimetry program developed for this work. Combining these data with measurements of the simultaneous movements of the plasmodia revealed a simple physical mechanism of locomotion. The shuttle streaming of the protoplasm was not truly symmetric due to the peristalsis-like movements of the plasmodium. This asymmetry meant that the transport capacity of the stream was not equal in both directions, and a net forward displacement of the center of gravity resulted. The generality of this as a mechanism for amoeboid locomotion is discussed.     

Entamoeba motility: dynamics of cytoplasmic streaming, locomotion and translocation of surface-bound particles, and organization of the actin cytoskeleton in Entamoeba invadens.

The dynamics of cytoplasmic streaming, retrograde translocation of externally bound particles and locomotion by Entamoeba invadens were compared. Locomoting amoebae were monopodial, exhibited fountain flow cytoplasmic streaming and translocated externally bound erythrocytes to the rear of cells. The rates of rearward flow of peripheral cytoplasmic vacuoles and of the externally bound particles were equal to the rate of cell forward locomotion. Rhodamine-phalloidin staining revealed a distinct cortical polymerized actin cytoskelton. This was least evident about the periphery of the advancing pseudopod, increased in density toward the rear of the cell and was most concentrated in the uroid. A monoclonal anti-eucaryotic actin antibody, which recognized monomeric Entamoeba actin on immunoblots, stained trophozoites by indirect immunofluorescence throughout the cytoplasm, but not in the cortical regions stained by rhodamine-phalloidin. This and other evidence implied that the antibody recognized only unpolymerized actin in Entamoeba. We propose that locomotion, cytoplasmic streaming and translocation of externally bound particles are driven by a common actin-based mechanism in Entamoeba, possibly involving retrograde cortical actin flow and recycling.     

Mechanics and control of the cytoskeleton in Amoeba proteus.

Many models of the cytoskeletal motility of Amoeba proteus can be formulated in terms of the theory of reactive interpenetrating flow (Dembo and Harlow, 1986). We have devised numerical methodology for testing such models against the phenomenon of steady axisymmetric fountain flow. The simplest workable scheme revealed by such tests (the minimal model) is the main preoccupation of this study. All parameters of the minimal model are determined from available data. Using these parameters the model quantitatively accounts for the self assembly of the cytoskeleton of A. proteus: for the formation and detailed morphology of the endoplasmic channel, the ectoplasmic tube, the uropod, the plasma gel sheet, and the hyaline cap. The model accounts for the kinematics of the cytoskeleton: the detailed velocity field of the forward flow of the endoplasm, the contraction of the ectoplasmic tube, and the inversion of the flow in the fountain zone. The model also gives a satisfactory account of measurements of pressure gradients, measurements of heat dissipation, and measurements of the output of useful work by amoeba. Finally, the model suggests a very promising (but still hypothetical) continuum formulation of the free boundary problem of amoeboid motion. by balancing normal forces on the plasma membrane as closely as possible, the minimal model is able to predict the turgor pressure and surface tension of A. proteus. Several dynamical factors are crucial to the success of the minimal model and are likely to be general features of cytoskeletal mechanics and control in amoeboid cells. These are: a constitutive law for the viscosity of the contractile network that includes an automatic process of gelation as the network density gets large; a very vigorous cycle of network polymerization and depolymerization (in the case of A. proteus, the time constant for this reaction is approximately 12 s); control of network contractility by a diffusible factor (probably calcium ion); and control of the adhesive interaction between the cytoskeleton and the inner surface of the plasma membrane.     

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.     

Cell migration by graded attachment to substrates and contraction

The migration of fibroblastic cells in vitro involves the extension of lamellipodia, attachment of the cytoskeleton through the plasma membrane to the extracellular matrix, and the generation of force that pulls attachments rearward and the cell forward. Bulk flow of membrane or lipid relative to the cell outline cannot be detected; however, crosslinked glycoproteins attached to the cytoskeleton move rearward and diffusing particles are driven forward by a motor mechanism. The leading edge is the preferential site for the cytoskeleton to attach to crosslinked glycoproteins including integrins. Force for moving the cell forward can be generated either by a cortical contraction acting as a net to pull the endoplasm forward or by motors at the boundary of the endoplasm and ectoplasm pulling on the cortical actin. As the cortical actin is anchored to the external matrix more strongly at the front of the cell than at the rear, contraction will pull the cell forward. Such a model has important implications for the nature of the glycoprotein attachments to the cytoskeleton and the regional differences in membrane structure.     

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.     

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.     

Localised depletion of polymerised actin at the front of Walker carcinosarcoma cells increases the speed of locomotion

Spontaneously migrating Walker carcinosarcoma cells usually form lamellipodia at the front. Combined treatment with 10(-5)M colchicine and 10(-7)M latrunculin A produces large defects in the cortical F-actin layer at the leading front and suppresses lamellipodia. However, the cortical actin layer at the rear is intact and shows myosin IIA accumulation. These cells, showing no or little detectable cortical F-actin at the front and no morphologically recognisable protrusions, migrate faster than control cells with lamellipodia and an intact cortical actin layer. This documents that the cortical actin layer or actin-powered force generation at the front is redundant for locomotion. Colchicine and latrunculin A have synergistic effects in compromising the cortical layer at the front and in increasing the speed of locomotion, but antagonistic effects on the relative amount of F-actin per cell. Colchicine but not latrunculin A, can increase the proportion of polarised and locomoting cells under appropriate conditions. Locomotion and polarity of cells treated with latrunculin A and colchicine is inhibited at latrunculin A concentrations >10(-7)M, by the myosin inhibitor BDM or the ROCK inhibitor Y-27632. Colchicine and Y-27632 have antagonistic effects on polarity and the speed of locomoting cells. The data show that locomotion of metazoan cells, which normally form lamellipodia, can be driven by actomyosin contraction behind the front (cell body, uropod). They are best compatible with a cortical contraction/frontal expansion model, but they are not compatible with models implying that actin polymerisation or actomyosin contraction at the front drive locomotion of the cells studied.     

Pinocytosis and locomotion of amoebae. XIX. Immunocytochemical demonstration of actin and myosin in Amoeba proteus

The spatial distribution of cytoplasmic actin and myosin in 1. normal locomoting, 2. immobilized, and 3. pinocytosing Amoeba proteus was demonstrated by indirect immunofluorescence microscopy. In orthotactic and polytactic cells fixed during normal locomotion actin is mainly located in a cortical layer delineating the granuloplasm from the peripheral hyaloplasm. In cell areas lacking a hyaloplasmic sheet the actin layer immediately borders the plasma membrane. The amount of actin within the continuous layer seems to increase from the advancing front to the middle cell region and to decrease again toward the uroid. The distribution of myosin is largely congruent to the display of actin, with the exception that the myosin-based fluorescence of the cortical layer gradually increases from the front to the uroid. A considerable amount of actin and myosin is also distributed around the nucleus and the contractile vacuole. In immobilized cells contracted by the external application of 10(-4)M procaine hydrochloride the cortical layer distinctly increases in thickness. In contrast to normal locomoting cells actin and myosin show a uniform distribution within the cell cortex along the entire surface. In pinocytosing cells, up to three cortical layers conspicuously rich in actin are produced during the process of channel formation. One of these layers is located in close proximity to the plasma membrane of the pinocytotic channels and the vacuoles. The immunocytochemical results are discussed with respect to earlier observations on the distribution of actin and myosin in Amoeba proteus as obtained by other methods.     

Velocity measurements of particulate neuroplasmic flow in organized mammalian CNS tissue cultures

The movements of spherical particles in the range of 0.3 to 0.8 μm diameter within neurities of cultured embryonic mouse spinal cord fragments were observed and recorded by means of Nomarski optics and time-lapse photocinematography at high power. Particulate movements were measured by projecting the motion pictures onto a calibrated screen and recording the distances moved with time of linearly moving particles and making note of the direction (toward or away from the neuron soma) of movement. In all, 128 particles were measured in six cultures. These measurements were taken away from the neuron soma near the periphery of the neurites. Eighty-three particles were noted to be moving toward the neuron at a mean velocity of 1.03 ± 0.38 (SD) μm/sec while 45 anterograde moving particles were noted to move at 1.07 ± 0.62 (SD) μm/sec. Statistical analysis of these velocities revealed no significant difference between them. Particles which were elongated and probably represented mitochondria moved more sluggishly and could not be measured accurately by the techniques employed. It appeared the spherical particles moving in a retrograde direction originated at the neurite tip apparently by pinocytosis. There was a suggestion that anterograde flow and retrograde flow may have been affected unequally by factors which develop in the observation chamber over a period of 2 hr or more. The most likely factor responsible was probably hypoxia.     

Velocity measurements of particulate neuroplasmic flow in organized mammalian CNS tissue cultures.

The movements of spherical particles in the range of 0.3 to 0.8 mum diameter within neurities of cultured embryonic mouse spinal cord fragments were observed and recorded by means of Nomarski optics and time-lapse photocinematography at high power. Particulate movements were measured by projecting the motion pictures onto a calibrated screen and recording the distances moved with time of linearly moving particles and making note of the direction (toward or away from the neuron soma) of movement. In all, 128 particles were measured in six cultures. These measurements were taken away from the neuron soma near the periphery of the neurites. Eight-three particles were noted to be moving toward the neuron at a mean velocity of 1.03 +/- 0.38 (SD) mum/sec while 45 anterograde moving particles were noted to move at 1.07 +/- 0.62 (SD) mum/sec. Statistical analysis of these veolcities revealed no significant difference between them. Particles which were elongated and probably represented mitochondria moved more sluggishly and could not be measured accurately by the techniques employed. It appeared the spherical particles moving in a retrograde direction originated at the neurite tip apparently by pinocytosis. There was a suggestion that anterograde flow and retrograde flow may have been affected unequally by factors which develop in the observation chamber over a period of 2 hr or more. The most likely factor responsible was probably hypoxia.     

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.