Influence of extremely low frequency electromagnetic fields on the swimming behavior of ciliates.

Different species of ciliates (Paramecium biaurelia, Loxodes striatus, Tetrahymena thermophila) have been taken as model systems to study the effects of extremely low-frequency electromagnetic fields (50 Hz, 0.5-2.0 mT) on the cellular level. A dose-dependent increase in the mean swimming velocity and a decrease in the linearity of cell tracks were observed in all wild-type cells. In contrast, field-exposure did not increase the number of directional turns of the Paramecium tetraurelia pawn mutant (d4-500r), which is characterized by defective Ca2+-channels. The described changes indicate a direct effect of low frequency electromagnetic fields on the transport mechanisms of the cell membrane for ions controlling the motile activity of cilia.

Induced pinocytosis and endosomal pathways in Amoeba proteus.

The mode of endosome formation and processing was studied in Amoeba proteus by using bovine serum albumin (BSA) either coupled to the fluorochrome TRITC or to 12 nm gold particles (Au(12)) as a pinocytosis inducer. The intraendosomal modifications of BSA-TRITC and BSA-Au(12) start with the separation of the ligand complex from receptor sites of the endosomal membrane obviously caused by acidification (1-15 min). Subsequently, the delivery with lysosomal enzymes and the disintegration of the ligand complex into individual components (BSA and Au(12)) occurs (15-30 min). The intracytotic transport of the ligand components to distinct cellular compartments leads to separate endosome populations with a common fate, i.e. fusion with the cell membrane and exocytosis of the lysosomal content. However, a certain amount of vesicles containing the hydrolyzable ligand BSA escapes exocytosis and is delivered to the digestive apparatus thus contributing to the nutrient pool of the cell (30-40 min). The application of antibodies against human or Tetrahymena digestive enzymes resulted in the detection of a 22 kD protein which shows a distinct cross-reactivity with anti-cathepsin D. The protein is localized in the Golgi apparatus, in small vesicles and in secondary lysosomes containing ingested material. In general, induced pinocytosis can mainly be viewed as a survival mechanism developed by A. proteus to overcome extracellular stress situations. The consecutive endocytotic pathways involve the lysosomal system as well as some other intracellular compartments and are comparable to the situation in higher organisms.

Studies on microplasmodia of Physarum polycephalum: VIII. Visualization of a membrane skeleton and characterization of membrane proteins.

Small plasmodial fragments derived from the acellular slime mold Physarum polycephalum were used to demonstrate a functional relationship between ultrastructural changes of the membrane-cytoskeleton complex and rhythmic activities of the motive force generating system. During contraction the microfilament layer detaches from the plasma membrane and moves inwards thus causing syneresis of central granuloplasm from peripheral hyaloplasm. Simultaneously, on the internal face of the plasma membrane a more or less regular two-dimensional network of short filaments and small particles becomes visible resembling the spectrin membrane skeleton of erythrocytes. With incipient relaxation the contracted microfilament system disintegrates and a new layer is formed at the internal cell surface. Biochemical studies on isolated plasma membranes by gel-electrophoresis revealed three distinct bands of 29, 38 and 40 kD as well as some other conspicuous polypeptides such as actin, myosin and, especially, a high molecular weight actin-binding protein (HABP) of 230 kD. Moreover, a 22 kD component was recognized as an integral membrane glycoprotein by Triton X-114 extraction and Con A-peroxidase staining. It is discussed whether the 230 kD and 22 kD proteins are comparable to spectrin and the actin-binding protein ponticulin from Dictyostelium, respectively.

Visualization and measurement of calcium transients in Amoeba proteus by fura-2 fluorescence.

A fura-2 microspectrofluorimeter was used to visualize and measure intracellular calcium transients in normal locomoting and experimentally treated Amoeba proteus. The results show that subcellular heterogeneities of cytosolic free calcium, [Ca2+]i, correlate in time and distribution with characteristic patterns of protoplasmic streaming and ameboid movement. In detail, calcium ions have a dual effect by regulating both the contractile activities of the actomyosin cortex and the rheological properties of the cytoplasmic matrix. A high resting [Ca2+]i of 1.5 to 2.0 x 10(-7) M in the uroid region or in retracting pseudopodia is associated with the transformation of rigid ectoplasmic gel into fluid endoplasmic sol, and a low [Ca2+]i of 10(-9) to 10(-8) M in the front region or in extending pseudopodia with the re-transformation of endoplasmic sol into ectoplasmic gel. Locally increased peripheral [Ca2+]i accumulations higher than 10(-7) M are also observed at places where the actomyosin cortex is known to generate motive force by contraction, i.e., in the intermediate region of orthotactic amebas or in large pseudopodia of polytactic cells. External application of 30 mM KCl abolishes the intracellular Ca2+ gradient such that [Ca2+]i attains a uniform distribution and a maximum concentration of 2 x 10(-7) M; as a consequence, cells can show a transient loss of their locomotor activity and polarity by undergoing spherulation and total contraction. On the other hand, high external Ca2+ concentrations in the range of 100 mM stabilize the bipolar cellular organization, enhance the movement velocity and induce the propagation of Ca2+ waves repeatedly running from the uroid to the front region. The significance of external ions for signal transmission and the control of dynamic activities as well as the origin and fate of calcium participating in the observed transients are discussed.

The role of microtubules for the movement of mitochondria in pinacocytes of fresh-water sponges (Spongillidae, Porifera).

In pinacocytes around the base of fresh-water sponges (Spongillidae) mitochondria move over long, straight paths, at an average speed of 1.3 micron/s. The application of colcemid (10(-4) M) has a distinct influence on mitochondrial movement: after 45 min the paths are dramatically shortened, after 90 min the periphery is devoid of mitochondria, and after 180 min all the mitochondria have aggregated in the perinuclear region. Three hours upon removal of colcemid, most mitochondria start to migrate away from the nuclear region along new paths and reach the boundary of the cell after 4 h of reculturing. These dynamics were analyzed in serial video frames. Immunocytochemical staining of comparable pinacocytes with a monoclonal tubulin-antibody reveals a characteristic system of unbranched, radially arranged microtubules extending from the nucleus to the boundary of the cell. After the application of colcemid (10(-4) M), progressively fewer and shorter microtubules are labeled. Four hours after removal of colcemid, the microtubular system is restored. The results of the present paper imply that mitochondrial movement in pinacocytes is dependent on the existence of an intact microtubular system.

Dynamics of the cytoskeleton in Amoeba proteus. III. Influence of microinjected antibodies on the organization and function of the microfilament system.

Affinity-purified antibodies against actin, myosin, alpha-actinin and vinculin cross-reacted with corresponding proteins from Amoeba proteus in immunoblotting experiments. Antibody staining of cells fixed during locomotion revealed different distribution patterns with a local concentration of anti-actin in the intermediate and of anti-myosin in the uroid region. Anti-alpha-actinin labeled a thin layer at the internal face of the plasma membrane, whereas anti-vinculin was distinctly concentrated at the base of advancing pseudopodia. Microinjection of different control solutions or antibodies against actin, myosin and alpha-actinin neither influenced the normal morphology and motile activity of amoebae nor changed the cellular distribution pattern of complementary antigens. However, antibodies against vinculin disorganized controlled locomotion and altered the spatial morphology of the microfilament system as well as the localization of the vinculin antigen thus pointing to a function of this protein in adhesion and locomotion of A. proteus. The results of the present paper show similarities to observations on mammalian tissue culture cells.

High temperature-induced changes in the organization of the microfilament system and cell membrane activity in Amoeba proteus.

The response of Amoeba proteus to heating from 22°C to 45°C starts with the inhibition of locomotion when the temperature of the culture medium reaches about 32°C. All following morphological reactions which lead to the formation of spherical forms result from a centripetal contraction of the whole cell body along the former axis of migration. This contraction and changes in shape are closely correlated to alterations in the organization of the microfilament system and the cell membrane behaviour. During the initial phase of contraction, the cells first attain an irregular shape with a heavily folded surface and form thick plaques of microfilaments at distinct places beneath the cell membrane. Later, the filamentous plaques disappear probably as a consequence of further contraction and eventual destruction of the system. Destabilization and an increased fluidity of the cell membrane as induced by higher temperatures as well as controlled shifts in the equilibrium between actin assembly and disassembly and the increased synthesis of actin binding proteins are discussed as possible reasons for the described transformations.

Induced pinocytosis and cytoskeletal organization in Amoeba proteus - a combined fluorescence and electron microscopic study.

Induced pinocytosis was studied in Amoeba proteus by combining fluorescence microscopy, low-light-level-TV intensification, image processing and electron microscopy. After external incubation of living cells with fluorochromed bovine serum albumin (BSA-TRITC) or a complex of colloidal gold and fluorochromed bovine serum albumin (Au(12)-BSA-TRITC) the fate of internalized material was followed for more than 2 h. Endosomes containing the ingested marker/membrane receptor-complex gather rapidly (15-30 min) within the uroid region and fuse successively to one large secondary endosome which either segregates its content into the external medium by exocytosis or is pinched off with some surrounding cytoplasm from the cell body. In cases where endosome-fusion does not occur, the distinct vacuoles release their content exocytotically in a progressive fashion. The fluorescence and electron microscopic results are in agreement with the in vivo investigations and support the suggestion that the microfilament system is responsible for the generation of motive force necessary to promote endocytosis, endosome gathering, fusion, sequestration and exocytosis.

Sequestration of microinjected molecular probes from the cytoplasm of Amoeba proteus.

The intracellular fate of different molecular probes (TRITC, TRITC-BSA, TRITC-BSA-Au(16)) microinjected into Amoeba proteus was analyzed in living and fixed cells with the fluorescence and electron microscope, respectively. Immediately after microinjection all of the probes distribute randomly without impeding the normal movement behavior or cellular morphology. However, within 45 min numerous aggregates appear in the cytosol which measure 0.25-1 µm in diameter and consist of the condensed molecular probe as well as an unknown homogeneous material. Larger aggregates are then encircled by elements of the rough endoplasmic reticulum and autophagosomes are formed. Later, these autophagosomes fuse with pre-existing lysosomes and their content is finally released by exocytosis. About 4-6 days after microinjection the molecular probes are completely sequestered from the cytoplasm. Quantitative evaluations have shown that the mode and rate of autophagosome formation is clearly influenced by the biochemical properties, the intracellular concentration and the microinjected volume of the molecular probes. Uncoupled TRITC is more slowly and TRITC-BSA-Au(16) more rapidly sequestered from the cytoplasm than TRITC-BSA. High intracellular BSA-concentrations accelerate the rate of autophagosome formation, whereas a corresponding increase of the TRITC-concentration exhibits the contrary effect. Under constant fluorochrome and protein conditions the total number of vacuoles formed increases with the injected volume. The results of the present paper demonstrate that Amoeba proteus has developed a specific autophagosomal sequestration mechanism which is influenced by both, qualitative and quantitative differences of microinjected molecular probes.

Pinocytosis and locomotion of amoebae: XVII. Influence of different cations on induced pinocytosis in Amoeba proteus.

The influence of various monovalent (Na(+), K(+), Li(+)), divalent (Ca(2+), Sr(2+), Mn(2+)), trivalent (La(3+), In(3+), Ta(3+)), and polyvalent cations (egg albumin) on the pinocytotic activity of starved Amoeba proteus was studied by a modified channel-counting method. Accordingly, a distinct stimulation of pinocytosis is induced by all substances in the following order of intensity: Mn(2+) < Ca(2+) < Sr(2+) < Li(+) < K(+) < Na(+) < Ta(3+) < In(3+) < La(3+) < egg albumin. Equimolar mixtures of different cations exhibit a simple additive influence on the induction capacity with the exception of Ca(2+) and Mn(2+) which both inhibit pinocytosis of other inorganic or organic ions at mM-concentrations (1-150 mM); on the other hand, experiments using µM-concentrations (10-0.001 µm) delivered no perceptible effect of low external Ca(2+) levels on induced pinocytosis. Independent of the nature or substantial composition of the induction solution pinocytotic activity is also suppressed in a linear way beyond total molarities of 150-200 µM and completely restrained at concentrations of 600 mM. Specific ionophors (A 23187, valinomycin) and inhibitors (D-600, isoptin hydrochloride, amilorid, tetraethylamonium chloride) of ionic transport increase and decrease the rate of Ca(2+)- and, to a lower extend, Na(+)- or K(+)-induced pinocytosis, respectively. In this connection, the general significance of endogeneous and exogeneous calcium for the control of membrane flow and actomyosin contraction is discussed.

Chemically induced changes in the morphology, dynamic activity and cytoskeletal organization of Physarum cell fragments.

Spherical cell fragments derived from Physarum polycephalum by caffeine-treatment were used as an experimental system to investigate the influence of 15 externally applied substances on the general morphology, motile behavior and cytoskeletal organization of the acellular slime mold. In comparison to controls, the most obvious changes observed after chemical stimulation proved to be cytokinetic activities, ameboid-like movement phenomena, intense cell surface dynamics and formation of cytoplasmic actin fibrils. The results demonstrate the high adaptability of the microfilament system in Physarum even when subjected to extreme conditions in the external environment.

Studies on microplasmodia of Physarum polycephalum. VII. Adhesion-dependent changes in the organization of the fibrillar actin system.

Axenically-grown microplasmodia of the acellular slime mold Physarum polycephalum were used to study adhesion-dependent changes in the spatial organization of the cytoplasmic microfilament system. Results obtained by light- and electron microscopical techniques demonstrate the presence of a membrane-bound filament cortex in all microplasmodia, and the expression of additional cytoplasmic fibrils in specimens with tight contact to a substratum. The fibrils partly terminate in focal adhesion-sites and rather seem to serve a cytoskeletal than a contractile function.

Studies on microplasmodia of Physarum polycephalum : VI. Functional analysis of a cortical and fibrillar actin system by use of fluorescent-analog cytochemistry.

Fluorescently labeled actin (TRITC-G-actin) and heavy meromyosin (TRITC-HMM) derived from skeletal muscle and injected into microplasmodia of the acellular slime mold Physarum polycephalum were used to analyze the function of a cortical and fibrillar actin system in living specimens. The plasma membrane-attached cortical system can be labeled with TRITC-G-actin as well as with TRITC-HMM and visualized as a continuous sheath along the entire cell surface. Long-term experiments over time periods of several hours in conjunction with digital grey-value evaluations revealed that changes in the intensity of the fluorescent signal, as caused by alternative contraction and relaxation cycles of the cortical system, are distinctly correlated with periodic changes in the volume and shuttle streaming activity of the microplasmodia. The fibrillar actin system extending through the cytoplasmic matrix can be labeled only with TRITC-HMM. Formation and disappearance of fibrils were found to take place during relaxation and contraction of the cortical system, respectively. Results of the present paper indicate that the cortical actin system is mainly involved in motive force generation for alterations in cell surface morphology and locomotion activity, whereas the fibrillar actin system rather appears to maintain the mechanical stability of microplasmodia.

Distribution and dynamics of fluorochromed actin in living stages of Physarum polycephalum.

Isolated muscle and Physarum actins were labeled with various fluorochromes and microinjected into living stages of Physarum polycephalum (caffeine-droplets, endoplasmic drops, thin-spread macroplasmodia). Subsequent analysis of the intracellular redistribution by fluorescence microscopy, video-enhancement and digital image processing revealed RITC (rhodamineisothiocyanate) actin to be the most reliable molecular probe for the marking of microfilaments. In relaxed caffeine-droplets, the RITC-actin first diffuses randomly and then is locally incorporated into a thin cortical layer at the internal face of the plasma membrane. During Ca2+-induced contraction the fluorescent layer starts to detach from the plasma membrane, thus causing separation of central granuloplasm from peripheral hyaloplasm. Thin sections of both, relaxed and contracted specimens demonstrated that the RITC-actin layer in living droplets exactly coincides with a sheath of more or less oriented microfilaments. In contrast, RITC-bovine serum albumin (BSA) injected as control is excluded from those regions which show intense fluorescence with RITC-actin and the presence of an actin network with EM. Successful incorporation of the molecular probe into stages of Physarum polycephalum other than caffeine droplets was not yet achieved. The results obtained by fluorescent analog cytochemistry (FAC) are discussed with regard to the spatial organization of the actin system in acellular slime molds.

Preservation and phallotoxin-staining of the microfilament system in Amoeba proteus.

The spatial organization of the microfilament system as the main component of the cytoskeleton in Amoeba proteus was preserved by a glutaraldehyde-lysine-fixation and visualized with fluorescent phallotoxins (NBD- phallacidin , R-phalloidin). Results obtained by means of this method coincide exactly with observations gained from immunocytochemical, ultrastructural and molecular cytochemical studies, i.e., the microfilament system is mainly displayed beneath the cell membrane, at the hyalo - granuloplasmic border and around the cell nucleus. The preparation procedure employed is suitable for the rapid demonstration of cytoplasmic microfilaments in cells difficult to preserve by chemical fixation.

Intercellular coupling in frog heart muscle. Electrophysiological and morphological aspects.

Passive electrical parameters of bullfrog atrial trabeculae were measured in a single gap arrangement. Attention was focussed on the resistance of internal longitudinal pathway. The influence of external Ca2+ depletion was tested using EGTA as chelating agent. Morphometry of trabeculae, fine structure of junctional complexes, and distribution of membrane-bound Ca were investigated by light and electron microscopic methods. The specific internal resistance to longitudinal current flow was 523 omega cm with normal Ringer as perfusing fluid and 1140 omega cm in EGTA-containing solution. These values are considered to represent the sum of myoplasmic and junctional resistivity. Morphometrical studies indicated an interstitial space of 12%, a mean cell length of 358 micron, and a mean cell diameter of 3.2 micron. In freeze-fractured preparations junctional structures were observed in the form of "atypical gap junctions" consisting of 10 nm particles arranged in a circular or linear array. The number of gap junctions was estimated to range between 20 and 50/cell which is equivalent to a junctional area of 0.01 or 0.03% of total surface area. A mean number of 55 particles/gap junction was calculated. After 20 min of exposure to EGTA the majority of junctional complexes were converted to clusters; the number of particles/gap junction was not significantly altered. The fluorescent dye CTC was used as a probe for membrane-bound Ca of isolated living cells. In normal Ringer a strong fluorescence was seen at the cell surface and in different intracellular compartments. With EGTA both superficial and internal fluorescence disappeared completely. From a combination of electrical and morphometrical data the resistance of intercellular junctions was calculated. Under normal conditions the specific resistance of junctional membrane amounted to 0.4 omega cm2 and the resistance of an individual connection was of the order of 10(11) omega. With EGTA, the respective values were increased by about 230%. The mechanism underlying this depression of junctional conductance is not clear. It seems not related to a rise of cytoplasmic free Ca2+. The EGTA-induced increase in internal resistance was reflected by a decrease of the length constant of a bundle. The nature of "atypical gap junctions" and their relation to tight junctions are discussed. It is concluded that the junctions observed in frog atrial muscle are analogous to gap junctions of insect or mammalian cells in spite of the different size and arrangement of the particles. A theoretical model is presented for the electrical behaviour of a bundle in a single gap arrangement.(ABSTRACT TRUNCATED AT 400 WORDS)

Cytoplasmic actin patterns in Physarum as revealed by NBD-phallacidin staining.

Fluorescently labeled phallacidin, a F-actin specific drug, was used to demonstrate the morphological variety in the cytoskeletal actin pattern of thin-spread plasmodia of the acellular slime mould Physarum polycephalum. The patterns observed in phallacidin-stained specimens consisted of a polygonal network in the anterior region, and of longitudinal as well as helically twisted fibrils in plasmodial strands of the posterior region. These observations are in complete accordance with our recent results obtained on comparable plasmodia by immunofluorescence microscopy using specific antibodies against actin.

Immunocytochemistry of the acellular slime mould Physarum polycephalum. I. Preparation, morphology, and reliability of results concerning cytoplasmic actomyosin patterns in sandwiched plasmodia.

Small phaneroplasmodia of Physarum polycephalum migrate, under sandwich conditions between two agar sheets and a membrane of cellophane, as thin protoplasmic sheets. This method suitably simulates the situation in the natural habitat of acellular slime moulds; i.e. the narrow clefts of the forest soil. The highly differentiated system of cytoplasmic fibrils displayed under these conditions survives both long-term extraction with glycerol and fixation with methanol, procedures that remove the strong inherent autofluorescence, thus allowing the use of immunocytochemical studies. The complicated fibrillar system of sandwiched plasmodia consists of: (1) a membrane-associated cortical filament layer in the anterior region; (2) a more or less regular polygonal fibrillar network in the intermediate region; and (3) a helically twisted fibrillar system encircling endoplasmic pathways as well as isolated strands in the posterior region. So far, three different cytoskeletal proteins have been identified immunocytochemically as constituents of the fibrillar structures: actin, myosin and AM-protein (fragmin). No positive identification of alpha-actinin, filamin and tropomyosin was obtained using antibodies against vertebrate proteins. Electron microscopy of glycerol-extracted specimens treated with antibodies against actin and myosin revealed that the 6 nm filaments consist of actin, whereas the electron-dense material between single actin filaments appears to be myosin. The AM-protein modulating the polymer status of actin is located in all fibrillar structures.

Pinocytosis and locomotion of amoebae. XIX. Immunocytochemical demonstration of actin and myosin in Amoeba proteus.

The spatial distribution of cytoplasmic actin and myosin in 1. normal locomoting, 2. immobilized, and 3. pinocytosing Amoeba proteus was demonstrated by indirect immunofluorescence microscopy. In orthotactic and polytactic cells fixed during normal locomotion actin is mainly located in a cortical layer delineating the granuloplasm from the peripheral hyaloplasm. In cell areas lacking a hyaloplasmic sheet the actin layer immediately borders the plasma membrane. The amount of actin within the continuous layer seems to increase from the advancing front to the middle cell region and to decrease again toward the uroid. The distribution of myosin is largely congruent to the display of actin, with the exception that the myosin-based fluorescence of the cortical layer gradually increases from the front to the uroid. A considerable amount of actin and myosin is also distributed around the nucleus and the contractile vacuole. In immobilized cells contracted by the external application of 10(-4)M procaine hydrochloride the cortical layer distinctly increases in thickness. In contrast to normal locomoting cells actin and myosin show a uniform distribution within the cell cortex along the entire surface. In pinocytosing cells, up to three cortical layers conspicuously rich in actin are produced during the process of channel formation. One of these layers is located in close proximity to the plasma membrane of the pinocytotic channels and the vacuoles. The immunocytochemical results are discussed with respect to earlier observations on the distribution of actin and myosin in Amoeba proteus as obtained by other methods.