Minipodia and rosette contacts are adhesive organelles present in free-living amoebae.

Using scanning electron microscopy, Amoeba proteus cells migrating on the glass have been shown to develop dense coats of minipodia, which are discrete microprotrusions up to 8 microm long and approximately 0.5 microm across. They cover the middle-anterior area of the ventral cell surface, i.e. the region previously determined as the zone of most efficient adhesion of an amoeba to its substratum. Minipodia are sparse underneath the frontal zone and lacking from the tail region. In amoebae that adhere to the glass without moving, have just started moving, or show unstable motor polarity, minipodia are grouped in rosette contacts, cauliflower-like papillae composed of supporting platforms with crowns of minipodia emerging from them. Both structures abound with cytoskeletal F-actin, as shown by staining with fluorescein-conjugated phalloidin. Amoebae experimentally prevented from adhering to the substratum neither develop discrete minipodia nor rosette contacts.

Reversible changes in size of cell nuclei isolated from Amoeba proteus: role of the cytoskeleton.

Micrurgically isolated interphasal nuclei of Amoeba proteus, which preserve F-actin cytoskeletal shells on their surface, shrink after perfusion with imidazole buffer without ATP, and expand to about 200% of their cross-sectional area upon addition of pyrophosphate. These changes in size may be reproduced several times with the same nucleus. The shrunken nuclei are insensitive to the osmotic effects of sugars and distilled water, whereas the expanded ones react only to the distilled water, showing further swelling. The shrinking-expansion cycles are partially inhibited by cytochalasins. They are attributed to the state of actomyosin complex in the perinuclear cytoskeleton, which is supposed to be in the rigor state in the imidazole buffer without ATP, and to dissociate in the presence of pyrophosphate. Inflow of external medium to the nuclei during dissociation of the myosin from the perinuclear F-actin may be due to colloidal osmosis depending on other macromolecular components of the karyoplasm.

Surface coat shedding and axopodial movements in Actinophrys sol.

Dyes specific to the glycoproteins of the mucous coat (Alcian Blue and Ruthenium Red) and ligands cross-linking the surface receptors (γ-globulin and Concanavalin A) provoke detachment of the surface coat and shedding of mucus conglomerates outwards along axopodia in Actinophrys sol. Moreover, the two receptor-specific ligands induce rearrangement of axopodia into unipolar fan-like bundles. They arise by inclination of axopodia in one direction, usually before shedding of the surface coat. Then, the mucus is unidirectionally evacuated along the bundles. The coordinated polar reorientation of axopodia is faster than any other axopodial movement and manifests the dynamics of an active behaviour. However, its mechanism remains conjectural.

Dissociation of membrane-cortex contacts in the hyalospheres of Amoeba proteus exposed to light-shade differences.

The hyalospheres produced by a heat shock spontaneously separated successive sheets of the cortical actin layer from the plasma membrane and retracted them inward. This phenomenon was hampered or completely inhibited by 10(4) lux white light and restored in shade. The frequency of detaching the consecutive submembrane sheets was much higher in the shade than in full light. If the light-shade difference has been applied across a single hyalosphere, the detachment of cortical layer was initiated and continued in the shaded cell part. Sometimes it was followed by translocation of the hyaloplasm into the dark zone and a compensatory shift of the granuloplasmic core toward the bright area. Probably, the actin sheets which are detached in the frontal caps of normal locomoting amoebae react in the same way to positive or negative photic stimuli.

Dynamics of membrane-cortex contacts demonstrated in vivo in Amoeba proteus pretreated by heat.

The dissociation of membrane-cortex contact in the fronts of moving amoebae, which was earlier stated mainly in the fixed material is now demonstrated in vivo and cinematographically recorded in living cells pretreated for 15-30 minutes at 40°C. The cells round up and then, when they are cooled at room temperature, the cortex completely dissociates from the membrane and envelops the granuloplasm aggregated in the cell centre, whereas a hyaloplasmic ring, 40-50 µm broad, develops along the whole periphery. Such cells, called by us the hyalospheres, are viable and manifest various intracellular movements, and may eventually recover the capacity to locomote. New cortical layers are rebuilt under the cell membrane, periodically separated from it at 5-10 second intervals, and retracted as optically dense sheets across the hyaloplasmic ring toward the granuloplasmic core. Their separation may be spontaneous, but is strongly promoted by some agents increasing the membrane mobility, which in normal amoebae induce the formation of new fronts of locomotion. The formation of endocytotic invaginations, channels and vacuoles is also easily observed within the large and clear hyaline zone. Some new forms of them are described. The cyclic endocytotic activity is repeated at the steady frequency by the same spots at the cell surface. The hyalospheres may serve as simplified models to investigate in vivo the membrane-cortex interactions involved in the mechanisms of motor behaviour and endocytosis in amoebae.

Two-directional pattern of movements on the cell surface of Amoeba proteus.

Particles of latex, glass and precipitated Alcian Blue were studied cinematographically on the surface of migrating Amoeba proteus and in the surrounding medium. The majority of the attached and all unattached particles flow steadily forward in the direction of the endoplasmic streaming and cell locomotion. Flow on the surface is faster than in suspension. Some particles stuck on the membrane move backwards from the frontal region. This retrograde transport is slower than the anterograde flow, and the rate decreases further when the particles approach cell regions adhering to the substratum, accurately following the pattern of the withdrawal of ectoplasm in the same zone. Both movements coexist in the same region and retrograde particles may pass anterograde ones at a distance less than their diameter. Transition from forward flow to backward transport occurs just behind the frontal cap, where the new ectoplasm is formed. The anterograde movement is interpreted as reflecting the general forward flow of the laterally mobile fluid membrane components, which become added to the frontal surface of the locomoting cell; the retrograde movement as retraction of membrane components that, externally, are linked to the transported material and, on the cytoplasmic side, to the contractile microfilamentous layer, as is postulated for cap formation in tissue cells.

Functional interdependence of pseudopodia in Amoeba proteus stimulated by light-shade difference.

Polytactic cells of Amoeba proteus were exposed to localized photic stimulation. When a pseudopodium is stimulated to advance, by shading it, other pseudopodia are retracted. Activation of the shaded front is the primary response, and contraction of other fronts the secondary one. When a pseudopodium is inhibited by illuminating its frontal segment, or when it is allowed to enter the bright zone in the course of migration, it slows down and stops but its eventual retraction depends on the existence of other possible directions for the endoplasmic flow. Therefore, if other active pseudopodia are lacking, the front suppressed by light cannot retreat effectively until new fronts arise in other body regions kept in shade. In all experimental situations the development of new fronts or the activation of forward flow in lateral pseudopodia precedes the contraction of the former leading pseudopodium. Also the reversal of direction of the endoplasmic streaming begins at the new front, and then it gradually extends until it reaches the former front. The results confirm the interdependence of different pseudopodia in the same individual and they contradict the concept that pseudopodia behave as separate functional units. On the other hand, they indicate that formation of new pseudopodia should not be explained as a simple secondary effect of contraction of the older ones but, on the contrary, as a phenomenon that initiates the changes in the pattern of flow in amoeba. The general interpretation is based on this variant of the pressure-flow theory of amoeboid movement, which attributes the motive power to the contractile activity of the whole cell cortex and the steering role to events taking place in the front of the migrating cell.

Effects of localized photic stimulation on amoeboid movement and their theoretical implications.

The uroids, the posterior, middle and anterior segments of the intermediate body regions, and the frontal zones of Amoeba proteus were exposed, separately or in different combinations, to local increases or decreases of light intensity. In general, an increase of luminosity promotes contraction, and its decrease induces relaxation in any body region of amoeba. The contracting or relaxing effects of luminosity changes are detectable along the whole length of the cortical tube, including its most anterior part. The response induced by stimulation of the tip of an advancing pseudopodium is functionally opposed to the reaction elicited by the same stimulus acting on all other body regions. The uroidal retraction and frontal extension are accelerated by illumination any segment of the cortical tube, or by shading the frontal zone. They slow down when shade is applied to any part of the tube, or light to the front alone. The motor efficiency of the light-induced contraction is modified by the position of the simulated area in respect to the adhesion sites. Velocities of uroidal retraction and frontal extension depend (in the same degree as on the cortical contraction) on the availability of frontal openings accessible for the endoplasmic outflow from the cortical tube. Thus, the uroid accelerates when more fronts are formed, and an older advancing pseudopodium slows down when it has to compete with new fronts. It is concluded in general that the movement of amoeba depends on the contractile activity of the whole cell cortex which plays the motor role, and on the opening or obscuring the frontal breaches in the cortical envelope which perform the controlling and steering roles in locomotion.

Testing motor functions of the frontal zone in the locomotion of Amoeba proteus.

The hypothesis which attributes the motive power to the frontal zone has been tested by cinematographic analysis of the movements of amoeba, in which the front was either blocked by negative stimuli or destroyed. Partial inhibition and consecutive reorganization of the frontal activity by a beam of light had minor effect on the retraction of other body parts. Microsurgical destruction of the whole frontal zone had no effect on the functions of more posteriorly situated cell regions, which continue to contract and squeeze the endoplasm out into the external medium.

Testing steering functions of the frontal zone in the locomotion of Amoeba proteus.

Light and chemical stimuli were asymmetrically applied to the advancing front of amoeba without affecting any body region behind the frontal zone. Stimulation limited to the front alone was sufficient to control the frontal expansion and, as a further consequence, the locomotion and shape of the whole cell. Contracting factors applied locally to the front inhibited it, whereas the relaxing agents activated its expansion.

Plasmodium of Physarum polycephalum as a synchronous contractile system.

The contractile activity of veins and the rhythmicity of the frontal progress were photometrically recorded from cine-film, at numerous points across the whole plasmodium. Graphic analysis of the obtained curves demonstrates the existence of common contraction rhythm over the entire network, coincident to the expansion rhythm of the advancing front. It is suggested that the plasmodium of Physarum polycephalum represents an imperfectly synchronized monorhythmic contractile system.

Response to light-shade difference in anucleate and polynucleate specimens of Amoeba proteus.

The enucleated specimens of Amoeba proteus, the anucleate fragments, and the polynucleate individuals which all are capable of cortical contraction but not of locomotion, may be reactivated by the light-shade difference established across their body. Individual cells or fragments migrate toward the shade. The motory polarity and coordinated movement disappear immediately after cessation of the stimulus. The results are interpreted according to the earlier hypothesis that the necessary to maintain the motory polarity of amoebae. It is suggested that the anucleate and polynucleate specimens are incapable of coordinated movements when non-stimulated, because of a deficiency or an excess, respectively, of the regulatory relaxing factor secreted by the nucleus of Amoeba proteus.