Immunocytochemistry of the acellular slime mold Physarum polycephalum. II. Spatial organization of cytoplasmic actin.
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Actins↗
Biomedical subjects
W Stockem
Publications and source records attributed to W Stockem.
Cytoplasmic actomyosin fibrils after preservation with high pressure freezing.
The fine structure of the actomyosin system of Physarum polycephalum was investigated in vitrified specimens after applying a pressure of greater than 2.1 kbar and freezing rates of 500 to 5,000 degrees C/s. The frozen specimens were either freeze-substituted or freeze-fractured and compared with material processed according to conventional methods of freeze-etching preparation. Artifactual alterations, as seen in the form of destroyed areas of the cytoplasm after chemical fixation, were not observed after freeze-substitution. However, small ice crystals formed by recrystallization within most of the cytoplasmic actomyosin fibrils prevented a fine structural analysis. Such a destruction of the fibrillar fine structure was not found after freeze-etching. In replicas of deep-etched objects 10 nm-thick filaments were localized, which could be conclusively identified as F-actin. The actin filaments are located randomly in the peripheral cytoplasm forming the cell cortex. By the process of parallel aggregation, the filaments can be differentiated to fibrils. Thick myosin filaments were not observed. However, structures resembling cross bridges between single actin filaments suggest the existence of oligomeric myosin. The present investigation shows that, in addition to biomembranes, other cytoplasmic differentiations such as components of the groundplasm can be successfully demonstrated employing the deep-etching technique when the freezing methods are improved by avoiding freeze-protection pretreatments.
Actomyosin↗
Visualization of actin polymerization and depolymerization cycles during polyamine-induced cytokinesis in living Amoeba proteus.
Microinjection of spermine induces cytokinesis of Amoeba proteus. Within 30--60 s after spermine injection cells form one, or less commonly, two cleavage furrows and within the following 4--10 min the constrictions are completed. The resulting nucleated cell parts show normal streaming and locomotion, whereas the non-nucleated cell parts remain stationary and later degenerate. The intracellular distribution of fully polymerization-competent fluorescently labelled muscle actin was followed by image intensification. Double injection experiments initially using labelled action and 30 min later spermine revealed a ring-like structure of enhanced fluorescence corresponding to the constricting cleavage furrow. Immediately after cleavage was completed, the ring disappeared. Electron microscopy of cells fixed during spermine-induced cytokinesis showed numerous well aligned actin and myosin filaments in the developing cleavage furrow. These filaments are a specialized manifestation of the cell cortex. The results demonstrate that cycles of actin and myosin polymerization and depolymerization and the parallel alignment of preexisting filaments (crosslinking) represent a basic mechanism in the generation of the motive force during cytokinesis.
Actins↗
Studies on microplasmodia of Physarum polycephalum. I. Classification and locomotion behavior.
Depending on the conditions of the axenic shuttle culture, microplasmodia of the acellular slime mold Physarum polycephalum can be subdivided into three classes regarding fine structural organization and protoplasmic streaming activity: (1) spherical and rod-shaped types, (b) ameboid types, and (c) symmetrical types. In ameboid microplasmodia, the motive force for the irregular protoplasmic streaming activity is generated by alternative contraction and relaxation of a membrane-associated layer, morphologically consisting exclusively of thin filaments (probably actin). The protoplasm flows along a hydraulic pressure gradient produced by the filament layer within limited regions of the cell periphery. In dumbbell-shaped microplasmodia the motive force for the regular protoplasmic shuttle streaming between the two spherical heads is generated both by volume changes of the peripheral cell region (caused by the contractile activity of the membrane-associated filament layer), and by volume changes of the internal cell membrane invagination system (caused by fibrils attached to the basal region of the invaginations). The development from the unordered protoplasmic streaming pattern and less complicated fine structural organization in ameboid microplasmodia to the highly organized protoplasmic shuttle streaming and the more complicated morphology in dumbbell-shaped microplasmodia can be explained by intermediate stages. Whereas the motive force for the transport of smaller amounts of protoplasm can be generated by the exclusive action of a cortical filament layer, the existence of a filament cortex, the display of cytoplasmic fibrils, and the development of plasma membrane invaginations appear to be a necessary precondition for the transport of large amounts of protoplasm.
Cytoplasm↗
Organization and spatial arrangement of fluorescein-labeled native actin microinjected into normal locomoting and experimentally influenced Amoeba proteus.
Fully polymerization-competent fluorescein-labeled actin from skeletal muscle was microinjected into both normal moving and experimentally treated Amoeba proteus. Its intracellular distribution was followed by integral image intensification of the fluorescence on a television screen and compared with controls injected with rhodamine-labeled serum albumin. The labeled actin was incorporated into the endogenous actin pool and exhibited a characteristic redistribution depending on the cellular morphology. Increased amounts of labeled actin could be detected within a thin layer separating the hyalo- and granuloplasm or running immediately beneath the plasma membrane when hyaloplasmic regions were absent. The topography of the fluorescent layer demonstrated in living cells is in agreement with the cortical microfilament layer described ultrastructurally recently in corresponding cells. The combined results emphasize the important role of the cortical filament layer in both morphogenetic processes (e.g., hyalo-granuloplasm separation or changes in cell shape) and motive force generation for cytoplasmic streaming and amoeboid movement.
Actins↗
Pinocytosis and locomotion of amoebae. XV. Visualization of Ca++-dynamics by chlorotetracycline (CTC) fluorescence during induced pinocytosis in living Amoeba proteus.
The dynamics of Ca++ during induced pinocytosis were studied in Amoeba proteus using chlorotetracycline (CTC). The fluorescence of the Ca++ - CTC-complex was monitored by an image intensification system, which has certain advantages over standard equipment: (1) Living cells are not subjected to the damaging influence of intensive microscopic illumination, (2) fluorescent probes are not bleached during observation, and (3) the rapid dynamics of the Ca++ -fluxes can be recorded using short exposure times. The results demonstrate the existence of Ca++ bound to intracellular and extracellular sites of the cell membrane complex in normal locomoting and pinocytotic Amoeba proteus. The application of cations inducing pinocytosis causes a rapid decrease in the external CTC-fluorescence probably due to a release of Ca++ from the mucous layer. The degree of fluorescence intensity is correlated with the capacity of pinocytotic channel formation, i.e., the fluorescence decreases as the number of channels increases. During the phase of vesiculation a distinct fluorescence mainly restricted to the basal region of the channels is observed. Intracellular Ca++ was detected in close vicinity to the plasma membrane after both microinjection and external application of CTC. The internal CTC-fluorescence is slightly decreased during the induction phase of pinocytosis. The observations are in good agreement with previous results on the localization of Ca++ -binding sites at the plasma membrane of Amoeba proteus and demonstrate the important role of Ca++ -fluxes for the process of pinocytosis.
Amoeba↗
The influence of an actin-modulating protein (AM-protein) from Physarum polycephalum on the cell motility of Amoeba proteus.
An actin-modulating protein (AM-protein) isolated from the acellular slime mold Physarum polycephalum and microinjected into living Amoeba proteus causes characteristic changes in cell shape, locomotory behaviour, and organization of the microfilament system of the amebae. The peptide chain weight of the AM-protein, which binds to one actin molecule with high affinity thus forming a heterodimer, is 43 000. The heterodimer is a powerful inhibitor of actin polymerization, when added to G-actin. A 50% inhibition is already obtained at a weight ratio of 0.05 AM-protein/actin. The AM-protein heterodimer has only little effect on F-actin, which can be neglected under the experimental conditions used. The microinjected AM-protein transforms normal locomoting polytactic amebae into orthotactic cell models. The orthotactic forms are characterized by a polarized organization, i.e. a flattened advancing cell pole consisting of one large pseudopod showing clear suppression of further pseudopod formation, and a contracting uroid undergoing cellular budding processes by the formation and vesiculation of numerous small evaginations. The observed changes in morphology can be interpreted as a relaxation of the advancing cell region due to a significant reduction of the polymerizable actin pool. As the AM-protein heterodimer does not attack F-actin filaments or the interaction of actin and myosin the middle region and the uroid of the microinjected cells are able to perform contractions which result in an orthotactic moving pattern. In conjunction with control experiments using double injection of AM-protein and phalloidin our observations prove the important role of controlled actin polymerization and depolymerization processes for cytoplasmic streaming activity and cell locomotion in ameboid cells.
Amoeba↗
Influence of different freeze-fracture pretreatments on the fine structure of Physarum polycephalum. A freeze-fracture and freeze-substitution study.
The influence of different fixatives (glutaraldehyde, osmium, osmium/glutaraldehyde, and osmium/mercuric chloride) and freeze-protecting agents (glycerol and sucrose) on the fine-structural preservation of micro- and macroplasmodia of the acellular slime mold Physarum polycephalum was investigated in both freeze-substituted and freeze-fractured material. Glutaraldehyde fixation and subsequent infiltration with glycerol or sucrose caused severe destruction in the morphology of plasmodial strands and protoplasmic drops, whereas osmium- or osmium/mercuric chloride fixation prevented the formation of normal fracture planes running through the hydrophobic core of the plasma membranes. A short prefixation in a mixture of osmium/glutaraldehyde followed by postfixation in glutaraldehyde delivered the most satisfactory results in the preservation of the fine structure. For comparison, the distribution of integrated membrane particles (IMP) was analysed in freeze-fracture replicas of unfixed controls as well as glutaraldehyde- and osmium/glutaraldehyde-fixed specimens by evaluating the number of IMP per 1 micrometer 2 in two different plasmodial regions; in the peripheral plasmalemma and in the central plasmalemmal invaginations. In controls not receiving chemical pretreatment and in specimens fixed with osmium/glutaraldehyde, the central plasmalemmal invaginations showed a clearly reduced total amount of IMP (exoplasmic + protoplasmic fracture face: about 3100) as compared with the peripheral plasma membrane (about 3700). In addition both membrane systems were characterized by an asymmetrical distribution of IMP between the protoplasmic fracture face (PF) and the exoplasmic fracture face (EF): the PF:EF ratio (particle partition coefficient) in the peripheral plasma membrane is the same in controls and in osmium/glutaraldehyde-fixed specimens (2.4:1 and 2.5:1, respectively), whereas the PF:EF ratio in the central plasmalemmal invaginations is 1.5:1 in controls and 3.5:1 in fixed specimens. This shows that the membrane of the central plasmalemmal invaginations is more sensitive to chemical fixation than the peripheral plasmalemma. The results point to differences in the physiological properties and functions between the plasmalemma of the cell periphery and the plasmalemma of the invagination system.
Cell Membrane↗
Effects of the actin-binding protein DNAase I on cytoplasmic streaming and ultrastructure of Amoeba proteus. An attempt to explain amoeboid movement.
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.
Amoeba↗
Pinocytosis and locomotion of amoebae: XII. Dynamics and motive force generation during induced pinocytosis in A. proteus.
The mechanism of induced pinocytosis was investigated in Amoeba proteus by light and electron microscopy. The application of nine different inducing substances revealed that pinocytotic channel formation, elongation, vesiculation, shortening and disappearance are the result of the successive or simultaneous action of both traction and pressure forces, which are produced by the contractile activity of a plasma membrane-associated layer of filaments ranging from a few hundred nm to several micrometer in thickness. The initial phase of channel formation is caused by traction forces according to the membrane flow concept, whereas channel elongation and vesiculation mainly result from pressure forces in conjunction with the extrusion of small hyaline pseudopodia. Shortening and disappearance of the pinocytotic channels are brought about by local contractions of the cortical filament layer in the basal region of the hyaline pseudopodia. Experiments using latex beads as marker particles together with inducing substances show that a rapid membrane turnover duirng pinocytosis can be excluded, and that the plasma membrane slides as an entire structure over the underlying cytoplasm.
Amoeba↗
Studies on microplasmodia of Physarum polycephalum V: electrical activity of different types of microplasmodia and macroplasmodia.
Results obtained by three different electrophysiological methods delivered mainly oscillating potentials (independently of the type of organization of Physarum polycephalum) with an average period length of 1 min 21 sec. This value is in good statistical agreement with corresponding data for the periodical oscillation of the shuttle streaming (1 min 47 sec) and coincides also with the radial tensiometric contractile activity (1 min 34 sec). The investigation favours the hypothesis that ion fluxes across the cell membrane and perhaps between intracellular compartments may act as a trigger mechanism for a still unknown oscillator-system which controls cell movement phenomena in the acellular slime molds.
Cell Membrane↗
Cytoplasmic streaming in Amoeba proteus is inhibited by the actin-specific drug phalloidin.
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Amoeba↗
[Cytological and electrolyte-histochemical investigations on freeze-substituted amoebae and acellular slime molds (author's transl)].
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Amoeba↗
Fine structure of cytoplasmic crystal in Amoeba proteus.
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Amoeba↗
The influence of microinjected phalloidin on locomotion, protoplasmic streaming and cytoplasmic organization in Amoeba proteus and Physarum polycephalum.
Microinjected phalloidin induces both time and concentration-dependent changes in morphology and motility of amoebae and acellular slime moulds. In A. proteus injection of a 10(-3)M solution of the drug causes a separation of cortical hyaline plasma from central granular plasma. Simultaneously protoplasmic streaming and cellular locomotion are lost irreversibly. Lowering the concentration of phalloidin to 2 x 10(-4)M results in a reversible disturbance; amoebae recover after 30 to 60 minutes and show normal movement. In Ph. polycephalum the injection of a 10(-3)M solution of phalloidin into single veins induces a local gelation of the protoplasm followed by the separation of hyalo- and granuloplasm. In semi-thin and ultrathin sections the hyaline plasma regions contain a fine granular groundplasm rich in ribosomes but free of cellular organelles. The central granular plasma consists mainly of membrane-surrounded cellular compartments. The two morphologically distinct plasma regions are separated by a 0.5 to 1.0 micrometer layer of filamentous material. In A. proteus the filamentous layer is found shortly after phalloidin injection in close proximity to the plasma membrane, and consists of thin 5 to 6 nm filaments. With increasing time this layer contracts, separates from the inner plasma membrane and moves to the interior of the cell. During contraction thicker filaments with diameters of 10 to 30 nm and lengths of 300 to 500 nm are formed. The results indicate that the display and contraction of the phalloidin-induced filament layer can account for the changes observed in cellular movement and cytoplasmic organization. The resulting phenomena i.e. separation of hyaline plasma from granular plasma and changes in both the protoplasmic streaming pattern and locomotory activity of the cells, are discussed in terms of a general understanding of amoeboid movement.
Amoeba↗
[Cytochemical studies on intracellular digestion in the acellular slime mold Physarum confertum (author's transl)].
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Acid Phosphatase↗
[Preparation of biological specimens for the scanning electron microscope (author's transl)].
Three methods usually applied in preparing biological material for the scanning electron microscope were tested by the investigation of two species of amoebae with different content of water (Amoeba proteus, Vannella simplex). Air drying resulted in both the production of cell shrinkage and cell distortion. When the specimens were dryed from media with increasing vapour-pressure, more satisfactory preservation of surface structures could be obtained. The sequence of potency was: Ethanol, chloroform, isopentane, ethyl ether, freon 11 and freon 13. Short drying periods proved to be more favourable than long ones. Critical-point drying provided much better details of cell surface morphology in both amoebae species. However, some arteficial changes were still detectable as small breaks and destruction of the mucous layer. They must be attributed to the fixation and dehydration procedure. Freeze drying turned out to be superior to both air drying and critical-point drying. Specimens prepared by this method showed no visible differences in cell surface morphology compared to living cells. As a consequence of the relatively high content of water the preparation of A. proteus was more difficult than that of V. simplex.
Amoeba↗
Effects of induced pinocytotic activity and extreme temperatures on the morphology of Golgi bodies in Amoeba proteus.
The morphology of the Golgi apparatus of Amoeba proteus can be influenced by substances inducing pinocytotic activity as well as by extreme temperatures. During the ingestion of a solution of 0.5% egg white the number of Golgi bodies decreases from 100% measured in control cells to 82% measured in cells showing induced pinocytosis. Simultaneously the ratio of the surface area of the cisternae at the proximal face to that of the vesicles at the distal face of single dictyosomes remains constant (1.74-1.72). The decrease and increase of the temperature of the culture medium to 4 degrees C and 30 degrees C respectively, causes the disappearance of most of the dictyosomes. After keeping the cells for 3-10 h at these temperatures the number of Golgi bodies was only 5-10% of the controls. A continued treatment with cold or warm culture medium leads to a partial reorganization of dictyosomes. After 15 h the number of Golgi bodies counted per cell returned to 57% at 4 degrees C and 38% at 30 degrees C. The ratio of the surface area of the Golgi cisternae to the surface area of the Golgi vesicles also alters under the influence of extreme temperatures. The values measured after treating the cells for 3h, 4 h 10 h and 15 h at 4 degrees C and 30 degrees C amounted to 0.75, 0.85, 1.14 1.53 and 0.93, 0.38, 0.88, 1.60, respectively, compared to 1.72 of control amoebae. The different values of the ratio of the surface area of cisternae to that of vesicles indicate that there are strong morphological changes of single dictyosomes.
Amoeba↗