Andrzej Grębecki

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.

Bidirectional transport of extracellular material by the cell surface of locomoting Saccamoeba limax

Summary The movement of latex spheres and some other particles along a migrating S. limax has two components. The majority of particles flow forwards at the same rate as the cell locomotion rate or slightly faster. This anterograde transport is manifested by the particles which seem to be loosely attached to the cell surface and is transmitted even to those remaining in suspension. It is ectoplasm independent: the particles overtake the ectoplasmic granules and the outline of the cell contour. The second component, which may occur in the same time and in the same place is, on the contrary, ectoplasm-dependent. It is manifested only by the particles firmly attached to the surface (probably by adhesion), which move exactly in the same manner as the ectoplasmic layer on the opposite side of the membrane. Their transport is, therefore, always retrograde relatively to the oell position. In respect to the ground, they move backwards along the frontal fountain zone, are stationary at the intermediate cell-to-substratum attachment zone, and on the tail surface are transported forwards but slower than the cell locomotion rate. It is presumed that the first component relfects the flow of the fluid lipid fraction of the cell membrane, while the second the behaviour of surface receptors engaged in the adhesion of the particles, which are linked to the submembraneous contractile cell cortex.

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.

Locomotion of Saccamoeba limax

Summary The unidirectional locomotion of Saccamoeba limax is composed of a succession of pseudopodial cycles. Each new pseudopodium arises on the tip of its predecessor, as a new front with a hyaline cap. After the period of expansion, the old frontal edge is retracted backwards simultaneously with the initiation and expansion of a new front from one discrete site on the surface of the old one. This cycle is accompanied by establishing a new attachment to the substratum and its later detachment. Apparently monopodial Saccamoebae become typically polypodial when the attachment is lacking or restricted to the rear body end. All the ectoplasmic material is steadily retracted toward the attachment sites which were stable at the moment of observation. It produces the fountain movement in front of the attachment zone and tail retraction behind it. The fountain is very well expressed by the movements of cytoplasmic inclusions and of the outer cell contour. The tail retraction is never interrupted and much more steady than the progression of the front. Oscillations of the frontal velocity exceed in amplitude and precede in time the same oscillations in the withdrawing tail. The fronts respond negatively to the contracting stimulation by light and positively to the relaxing influence of shade. It is highly probable that the generalized cortical contraction theory of amoeboid movement applies to S. limax. According to this concept the contractility of the whole cell periphery generates a steady motive power, while a relative relaxation in the front controls the rate and direction of the endoplasmic flow and cell locomotion.

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.

RELATIONSHIP BETWEEN PINOCYTOSIS AND ADHESION INAMOEBA PROTEUS

Induction of pinocytosis in Amoeba proteus is independent of adhesion. It is manifested by non‐adhering floating specimens, as well as by amoebae moderately adhering and locomoting on the glass, or tightly attached to the polylysine‐coated substratum. The formation of pinocytotic rosettes results in de‐adhesion, at the beginning of pinocytosis on the glass, but at its end on the polylysine. It suggests an opposition between adhesion and cell shape transformation. Pinocytosis and adhesion are both inhibited, by an unknown mechanism, in the presence of gelatin either in the substratum or in solution.

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.

Induction of lobopodia and lamellipodia in a filopodial organism (Vampyrella lateritia)

Summary The normally filopodial motor system of Vampyrella lateritia was modified by chemical agents that promote cell adhesion and spreading and by mechanical stimuli. Large lobopodia arose by cell surface blebbing between the filopodia. The cortical cytoskeletal layers periodically detached from the plasma membrane at the growing tips of lobopodia and moved backwards. Combined chemical and mechanical stimuli sometimes resulted in the development of lamellipodia, which were either circular as characteristic of cell spreading, or intensely expanded in one direction as usual in locomotion. Lamellipodia arise from filopodia by their swelling and fusion. After retraction they leave behind a reconstructed filopodial system. In general, in Vampyrella as in some other motile cells, lobopodia arise and function independently of filopodia, whereas the lamellipodia and filopodia are related by their origin and may be transformed one into another.

Cortical Flow in Free-Living Amoebae

The cell cortex of large fresh-water amoebae is composed of the plasma membrane covered with 200 nm thick mucopolysaccharide coat and connected on the inner side with the cytoskeleton. Lateral movements of this complex or of its components are currently called cortical flow (Bray and White, 1988), although the cell cortex cannot be considered as fluid.