Direct membrane effects of morphine and endorphins on Amoeba proteus.

Morphine, leu-enkephalinamide, met-enkephalin, alpha-neoendorphin and its Arg8 1-8 fragment increase contractile vacuole output in the freshwater Amoeba proteus at 18 microM. Significant effects of leu-enkephalin and naloxone are obtained at 180 microM. All compounds have reached their maximal activity at 720 microM. Alpha-neoendorphin and leu-enkephalin are inactive in the presence of isotonic, non-penetration sucrose, hence these compounds increase plasma membrane permeability to water. Results from molecular modeling show a clear correlation of activity with amphiphilicity, charge distribution and general flexibility of molecules. We conclude that, like previously-studied vasopressin analogues and non-hormonal amphiphilic peptides, active opioids embed themselves into the Amoeba plasma membrane, disrupting the lipid bilayer and increasing its permeability. In our Amoeba system, naloxone, a general morphine-like inhibitor, blocks active opioids as well as a vasopressin analogue. Naloxone, being less active than other tested amphiphiles, acts as a membrane stabilizer, protecting the lipid bilayer against the disruption action of more active compounds.     

Virotoxins polymerize actin and induce membrane fragmentation in cytoplasmic preparations of Amoeba proteus.

Virotoxins and phalloidin are peptides that induce actin polymerization in vitro. We have compared the effect of five virotoxins and phalloidin on the ultrastructure of spread preparations of Amoeba proteus cytoplasm. Like phalloidin, the five virotoxins induce polymerization of cytoplasmic actin. Moreover, the virotoxins, but not phalloidin, induce membrane fragmentation in small spherical vesicles. We, therefore, conclude that these virotoxins may have another membrane-bound target besides actin.     

Effects of the actin-binding protein DNAase I on cytoplasmic streaming and ultrastructure of Amoeba proteus

Microinjection of DNAase I, which is known to form a specific complex with G-actin, induces characteristic changes in cytoplasmic streaming, locomotion and morphology of the contractile apparatus of A. proteus. Light microscopical studies show pronounced streaming originating from the uroid and/or the retracting pseudopods, which ceases 10--15 min after injection of DNAase I, at a time when ultrasctructural studies show that the actin filament system is very much reduced. These results suggest that a controlled reversible equilibrium between soluble and polymerized forms of actin is a necessary requirement for amoeboid movement. The topographic distribution of contractile filaments beneath the plasma membrane visualized by correlated light- and electron microscopy of DNAase I-injected cells establishes the importance of the membrane-bound filamentous layer for three major aspects of streaming: (1) Streaming originates by local contractions of a cell membrane-associated filament layer at the uroid and/or retracting pseudopods, creating a pressure flow. (2) This flow continues beneath the membrane, which is stabilized by filaments in the lateral regions between the posterior end, with a high hydrostatic pressure, and the anterior end, with a low hydrostatic pressure. (3) Pseudopods or extending areas are created by a local destabilization of the cell periphery caused by the separation of the filamentous layer from the plasma membrane.     

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.     

Timing of nucleolar DNA replication in Amoeba proteus.

Light- and electron-microscope autoradiography have been used to follow the incorporation of [3H]thymidine at different stages during the interphase of synchronously growing populations of Amoeba proteus. Two main patterns were found for tritiated thymidine incorporation, i.e. DNA synthesis. The major incorporation was in the central region of the nucleus, but a lesser degree of incorporation occurred in the nucleolar region. The bulk of this nucleolar DNA was found to be late replicating, i.e. it replicated during the G2 phase.     

Selective Effects of Enucleation and Transfer of Heterologous Nuclei on Cytoplasmic Organelles in Amoeba proteus*

SYNOPSIS. The ultrastructural changes in the cytoplasm of lethal hybrids obtained by nuclear transplantation between different strains of Amoeba proteus were compared with those of enucleated amebae. It was found that, whereas the Golgi complex and glycocalyx degenerated first in enucleated cells, mitochondria and endosymbiotes became abnormal first in the hybrids. The selective effects are attributed to the presence of nucleic acids in the mitochondria and endosymbiotes and hence to the different interactions they would have with the nuclear genome.     

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.     

Incompatibility during transplantation of nuclei in amoeba. IV. Transplantation compatibility in different strains of Amoeba proteus]

Seven laboratory Amoeba proteus strains of different origin were tested for their transplantation compatibility (i.e. viability of artificially produced heterokaryons) in all possible pair combinations. Incompatible combinations (14) as well as completely compatible ones (7) were found. Compatibility was distributed among strain combinations non-randomly. All the strains studied could be classified into three groups with respect to compatibility ("compatibility groups") involving 1, 2 and 4 strains, respectively. Within the third group any pair of strains was compatible, as well as both strains of the second group. On the contrary, any strains from different groups were always incompatible. Possible nature of the compatibility groups is discussed. It is suggested that they might be analogous to syngens of Ciliates.     

Use of cell enucleation in studying the stability of cytoplasmic organelles and cytoplasm organization

A study was made of the viability and ultrastructure of cytoplasts produced by enucleation of cytochalasin-induced A9 cells in suspension. These cytoplasts are in general as viable as cells enucleated in the monolayer. The organization of the cytoplasm, i.e. a specific distribution of cytoplasmic organelles, is conserved for at least 24 hours in the absence of the nucleus. This fact may reflect a high degree of autonomy of the cytoskeleton.     

Peptide stimulation of phagocytosis in Amoeba proteus

Summary Normal articular cartilages from the weightbearing areas of the femoral condyles of the knee joints of 11 patients (3–20 years old) and of 35 Schwarzkopf sheep (3 months to 2 years old) were studied using the electron microscope. The study has shown that the matrix of normal articular cartilage is not only composed of collagen fibrils and proteoglycans, but also contains two types of elastic system fibres. Small elastic fibres can be identified in the superficial and lower radiate zones of cartilage of man and sheep. Similar to elastic fibres in other tissues, they consist of a central amorphous core and are surrounded by aggregates of 10 nm microfibrils. Another type of elastic system fibres, oxytalan fibres, are found in the intermediate and upper radiate zones of the articular cartilage.