Lucyna Grebecka

Is actin involved in the nuclear division in Amoeba proteus

During semi‐open mitosis of Amoeba proteus the nuclear envelope is not dispersed and nucleus divides by fission. The presence of actin layer close to nuclear envelope was demonstrated in interphase and telophase nuclei of that amoeba stained with rhodamine labelled phalloidin. In telophase, an accumulation of actin arises in the space between the future daughter nuclei. It appears to be comparable with the contractile ring of dividing cells. This suggests that actin associated with the nuclear envelope of Amoeba proteus may be involved in final separation of the daughter nuclei, forming a constriction ring at the middle of dividing nucleus.

Reversal of motory polarity ofAmoeba proteus by suction

SummaryWhen 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.

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.

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.

ADHESION-DEPENDENT F-ACTIN PATTERN INAMOEBA PROTEUSAS A COMMON FEATURE OF AMOEBAE AND THE METAZOAN MOTILE CELLS

Adhesion and movement ofAmoeba proteusare both dependent on the appropriate arrangement of the F‐actin cytoskeleton and on the presence of the cell nucleus. In this study the F‐actin organization was examined by routine FITC‐phalloidin staining and confocal laser microscopy in intact amoebae and in their nucleated and anucleated fragments, at different levels of cell adherence to the substratum. In the adhering and migrating intact cells and nucleated cell fragments dot‐like aggregates of F‐actin are scattered over the ventral side at sites close to the substratum. In the case of de‐adhesion of nucleated specimens this pattern disappears and F‐actin is accumulated in the cell centre and/or dispersed in the cytoplasm. The same actin distribution, without ventral dots, is found in the anucleated fragments which usually fail to attach to the substratum. Re‐adhesion of anucleated fragments, induced by a modified substratum or spontaneous, is accompanied by restoration of actin dots at the lower cell side. It is concluded that: (1) adhering specimens ofA. proteusdisplay the same dot‐like actin pattern on the ventral cell side, as many metazoan motile cells; (2) organization or disorganization of this pattern may occur independently of the presence of the cell nucleus, under the control of cell adhesion to the substratum.

Effects of bivalent cations on the initiation of Na-induced pinocytosis inAmoeba proteus

SummaryEGTA in moderate concentrations, sufficient to remove all Ca2+ from the cell surface, blocks pinocytosis. But in higher concentrations of EGTA, which chelate also Mg2+, the pinocytosis reappears and is strongly enhanced. Simultaneous removal of both Ca and Mg ions by EDTA brings about only potentiating effect. Reintroduction of either Ca or Mg separately, demonstrates that Mg2+ is a powerful inhibitor of pinocytosis. The influence of chelators on the pinocytosis is attributed respectively to their selective or unspecific influence on both bivalent ions at the cell surface, without affecting the intracellular contraction mechanism.

Motory effects of perforating peripheral cell layers ofAmoeba proteus

SummaryPerforation 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.

Externally applied nucleus explantant orientates the locomotion of Amoeba proteus.

The nucleus of Amoeba proteus acts on the cell cortex maintaining the activity and position of the frontal zone. Its effects are analogous to those of external stimuli. Both factors probably promote the separation of cell membrane from the underlying filamentous layer. In that way the hydrodynamic gradient that directs the cytoplasmic streaming and cell migration is created in the cell. Cessation of external stimulation does not disturb the cell locomotion, because the nucleus still maintains the necessary configuration of the frontal cortex. Reciprocally, the removal of the nucleus does not immobilize the cell, provided a strong enough external motor stimulus is acting. Only when both factors are removed or when the remaining single factor is too weak, the amoeba becomes incapable of locomotion. The cell nucleus and external stimulus to some extent replace each other, but an externally applied nucleus can be effective only in concert with the internal one. It seems, therefore, that the nuclear motor factor is rather weak, probably sufficient for maintaining the existing locomotory polarity but not for creating a new one. One can suppose, consequently, that the presence of the nucleus in the amoeba cell enables it to react to weak stimuli and persevere the orientated movement.

Adhesion to the substratum improves the motility ofAmoeba proteus in the absence of a cell nucleus

SummaryAnucleated fragments ofAmoeba proteus obtained by dissection and kept on an untreated glass surface fail to adhere to this substratum, lose motor polarity, and stop moving, at least for several hours. If they are transferred after the operation to a highly adhesive surface (polylysine-coated glass), they adhere to the substratum, although locomotion is not spontaneously restored. However, after exposure to a light-shade difference along their body they start moving towards the shaded area and continue locomotion as long as the photic stimulus is acting. Disorganisation of the F-actin cytoskeleton of anucleated fragments was observed on the untreated glass but reorganization on the polylysine-coated surface. The anucleated fragments can show transient recovery of slight spontaneous motor activity and react promptly to external stimuli after up to several days on untreated glass. These intermittent activity periods are enabled by reconstruction of F-actin cytoskeleton in the anucleated fragments during their temporary adhesion to the glass. It is concluded that the injurious effect of cell nucleus removal on the locomotor capacity of amoebae can be compensated by the simultaneous enhancement of cell adhesion and application of a stimulus restoring the motor polarity of the cell. The compensation is achieved by cytoskeletal reorganization.