A. Grębecki

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

SummaryThe 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.

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

SummaryBehaviour 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.

Relative motion inAmoeba proteus in respect to the adhesion sites

SummaryThe 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.

Fine structure and distribution of contractile layers inAmoeba proteus preincubated at high temperature

SummaryUltrastructural and immunocytochemical studies allow the localization and identification of a microfilament cortex in heat-shockedAmoeba proteus at different stages of recovery to room temperature. Immediately after heating the cortex is in close contact with the cytoplasmic face of the plasma membrane; however, during cooling it detaches from the membrane and shifts toward the cell centre thus separating a region of peripheral hyaloplasm from central granuloplasm. After polymerization of a new submembrane cortex several detachment and reformation cycles rhythmically repeated for 2–3 hours until a multitude of stratified layers has been formed in the hyaloplasm.Electron micrographs reveal that the cortical layer at the plasma membrane is merely composed of a network of actin filaments, whereas the retracted contractile layers in the hyaloplasm and at the granuloplasmic border contain both, thick and thin filaments often arranged in bundles. The heat-shock induced activities of the microfilament cortex are based on the highly contractile properties of this system in conjunction with controlled displacements in the equilibrium between F- and G-actin.

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

SummaryThe 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.

Resumption of locomotion byAmoeba proteus readhering to different substrata

SummaryFloating 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.

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

SummaryThe 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.

Locomotion ofAmoeba proteus after standardizing its body shape

SummaryAmoeba 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°.

Movement of surface markers along the pinocytotic pseudopodia ofAmoeba proteus

SummaryThe 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.