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W Stockem

Publications and source records attributed to W Stockem.

At least 19 recordsLinked to original sources
PubMed1997

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.

Animals↗
PubMed1991

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.

Amoeba↗
PubMed1990

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.

Animals↗
PubMed1990

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.

Actin Cytoskeleton↗
PubMed1987

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.

Actin Cytoskeleton↗
PubMed1987

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.

Caffeine↗
PubMed1984

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.

Amanitins↗
PubMed1984

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.

Actins↗
PubMed1983

Dynamics of the cytoskeleton in Amoeba proteus. I. Redistribution of microinjected fluorescein-labeled actin during locomotion, immobilization and phagocytosis.

Fluorescein-labeled muscle actin was microinjected into Amoeba proteus and followed during intracellular redistribution by means of the image-intensification technique. The fully polymerization-competent protein becomes part of the endogenous actomyosin system undergoing dynamic changes over time periods of several hours. Single-frame analysis of long-term sequences enabled the direct demonstration of both the contractile activities and morphological transformations of microfilaments in normally locomoting, immobilized and phagocytozing specimens. In normally locomoting cells the filament layer undergoes continuous changes in spatial distribution depending on the actual pattern of cytoplasmic streaming and cell shape. The highest degree of differentiation is always maintained in the intermediate region between the front and the uroid, thus indicating this segment of the cortex to be the most important site in generating motive force for pseudopodium formation and ameboid movement. In immobilized cells contracted by the application of ruthenium red or relaxed by different anesthetics, the filament layer forms a continuous thick sheath beneath the cell surface or becomes completely disintegrated. In phagocytozing cells the local polymerization of actin at the tip of pseudopodia forming the food-cup and around the nascent phagosome points to a significant participation of the actomyosin system in the process of capturing and constricting prey organisms. Although our results provide clear evidence for the overall importance of motive force generation according to the hydraulic pressure theory, some motile phenomena exist in Amoeba proteus that cannot exclusively be explained by this mechanism.

Actins↗
PubMed1983

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)

Animals↗
PubMed1983

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.

Actins↗
PubMed1983

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.

Actins↗
PubMed1983

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.

Actins↗
PubMed1983

Immunocytochemistry of the acellular slime mold Physarum polycephalum. III. Distribution of myosin and the actin-modulating protein (fragmin) in sandwiched plasmodia.

The acellular slime mold Physarum forms very thin plasmodia when sandwiched between two agar sheets. After extraction with glycerol-containing buffers, suitable objects for immunofluorescence microscopy are obtained, and an analysis of the cytoskeletal and contractile system of Physarum becomes possible. Plasmodia were stained with antibodies against myosin and fragmin, a protein factor involved in actin filament length regulation. The microanatomy and topography of cellular structures containing these proteins were investigated at the light and electron microscopic levels. The patterns obtained with the two antibodies are closely related to those obtained with actin antibody [25]. In both cases the complex system of cytoplasmic fibrils is stained selectively. The fibrils form a more or less regular network in the advancing front zone with the fibrils being interconnected by focal nodes. In the posterior region of the plasmodium, where endoplasmic pathways and protoplasmic veins are differentiated, larger fibrils are detected, running obliquely or longitudinally to the veins. With both antibodies the fluorescent pattern of the fibrils is continuous without indications of periodic interruptions or striations, which would be expected in the case of sarcomere-like subunits. With anti-myosin unstained patches are frequently seen at or close to the nodes of the fibrillar network in the anterior region. The small lobopodia, which are rich in actin, are apparently not stained by the myosin antibody, a result similar to the situation in "ruffling edges¿ of cultured vertebrate cells. Electron microscopic investigations of antibody-labeled fibrils in embedded and sectioned plasmodia allow the identification of antibody molecules at specific sites along the fibrils with a different distribution pattern for each of the two antibodies.

Animals↗
PubMed1982

Spatial organization and fine structure of the cortical filament layer in normal locomoting Amoeba proteus.

The fine structural organization of a cortical filament layer in normal locomoting Amoeba proteus was demonstrated using improved fixation and embedding techniques. Best results were obtained after application of PIPES-buffered glutaraldehyde in connection with substances known to prevent the depolymerization of F-actin, followed by careful dehydration and freeze-substitution. The filament layer is continuous along the entire surface; it exhibits a varying thickness depending on the cell polarity, measuring several nm in advancing regions and 0.5-1 micron in retracting ones. Two different types of filaments are responsible for the construction of the layer: randomly distributed thin (actin) filaments forming an unordered meshwork beneath the plasma membrane, and thick (myosin) filaments mostly restricted to the uroid region in close association with F-actin. The cortical filament layer generates the motive force for amoeboid movement by contraction at posterior cell regions and induces a pressure flow that continues between the uroid with a high hydrostatic pressure and advancing pseudopodia with low one. The local destabilization of the cell surface as a precondition for the formation of pseudopodia is enabled by the detachment of the cortical filament layer from the plasma membrane. This results in morphological changes by the active separation of peripheral hyaloplasmic and central granuloplasmic regions.

Amoeba↗