Evidence for actin transformation during the contraction-relaxation cycle of cytoplasmic actomyosin: Cycle blockade by Phalloidin injection

1) The injection of a mushroom drug, Phalloidin (750 microgram -1 mg/ml), into the endoplasmic channel of Physarum veins induces an irreversible blockade of the intrinsic contraction-relaxation automaticity of the ectoplasmic tube wall, as measured by tensiometrical methods. 2) The morphological responses to Phalloidin injection include an increase and condensation of cytoplasmic actomyosin sheets bordering the plasmalemma invaginations within the ectoplasmic tube and a more pronounced dense layer of "groundplasm" in the cell cortex. This is in accordance with experiments with other cells as well as with Physarum. 3) The addition of marker particles to the injection solution revealed that the injected substances can be brought into direct contact with the contractile substrate, before newly formed membranes separate off the injection fluid. 4) Since Phalloidin irreversibly transforms oligomeric actin into a filamentous "Phalloidin-actin complex" and because this transformation does not hinder the actin in activating myosin ATPase, it is concluded that the contraction-relaxation cycle of cytoplasmic actomyosin in Physarum involves actin transformations. If these transformations are hindered, e.g. by Phalloidin, one stage and thereby the whole cycle is sustained which results in a blockade of the intrinsic contraction automaticity. 5) The functional importance of actin transformations in the congraction physiology of cytoplasmic actomyosins and cell motility phenomena is discussed.     

Induction of a plasmodial stage of Physarum without plasmalemma invaginations

Experimentally generated protoplasmic drops of Physarum show time-dependent differentiation processes, i.e. regeneration of plasmalemma, actomyosin fibrillogenesis and regeneration of the plasmalemma invagination system. According to Hatano (1970), caffeine treatment of drops results in a pinching off process of small translucent droplets in which specific effects of Ca++ on protoplasmic streaming phenomena were demonstrated. The light and electron microscopic investigation of the original drop reveal that the time-dependent differentiation processes, e.g. actomyosin fibrillogenesis, are not inhibited by caffeine. However, caffeine hinders the regeneration of the plasmalemma invaginations in the original drop (up to a drop age of 30--40 min). The experimental advantage of this stage of Physarum with full vitality, but without plasmalemma invaginations is discussed.     

Enrichment of fibrillar cytoplasmic actomyosin in protoplasmic strands of Physarum polycephalum for the production of cell-free models

Summary The treatment of isolated protoplasmic strands of Physarum polycephalum with 2.5% ethanol in a physioogical salt solution under isometric conditions induces the formation of a large amount of mostly longitudinally organized actomyosin fibrils in the endoplasmic channel, a region normally free of actomyosin fibrils. The quantity of fibrillogenesis as well as the concomitant force output during the induced contractures are dependent on the Ca^{+ +}- content and the temperature of the test solution. The method was developed to optimize the structure of the plasmodial strands before their subsequent transformation into cell-free models by permeabilization and extraction of the strands.Cryosections of plasmodial strands containing cytoplasmic actomyosin fibrils stained with fluorescently labeled phallotoxins offer a further assay for the study of their contraction physiology under cell-free conditions.     

Spatio-temporal analysis of contraction dependent surface movements in Physarum polycephalum (author's transl)

Plasmodial veins of Physarum polycephalum were investigated by combining cinematographical and tensiometrical methods. Veins remaining on their original growing substrate show characteristic surface movements resulting from an intrinsic contraction automaticity. Radial and longitudinal components of surface movements were registered simultaneously. Both contraction activities show identical frequencies, in contrast to results derived from experiments with isolated veins. There is only one genuine frequency and therefore one has not necessarily to suppose the existence of a cooperation of two oscillating systems underlying the rhythmic contraction phenomena. The results are discussed in respect to the basis of the contraction phenomena: the cytoplasmic actomyosin fibrils of Physarum and their function in motive force generation for protoplasmic streaming.     

Reactivation of cell-free models of endoplasmic drops from Physarum polycephalum after glycerol extraction at low ionic strength

The effect of calcium ions on the reactivation of cytoplasmic actomyosin contraction in cell-free models of endoplasmic drops from Physarum polycephalum after glycerol extraction at low ionic strength depends on the duration of the extraction procedure: Ca++ prevents contraction in 20-h extracted specimens, whereas after several days of extraction this Ca++-sensitivity is lost. These results indicate an inhibitory effect of Ca++ on cytoplasmic actomyosin contraction.     

Reactivation of cell-free models of Physarum plasmodia after myosin reconstitution

Thin-spread glycerol-extracted Physarum plasmodia were treated with N-ethylmaleimide (NEM) to block myosin-ATPase and contractility. After supplementing the models with purified plasmodial myosin, they could be reactivated and contracted upon addition of ATP. Fluorescently labeled actomyosin fibers ruptured during contraction, resulting in beaded or rod-like contraction centers. Glycerol-extracted plasmodia lose their negative Ca++-dependence during extraction. Reconstitution of NEM-treated models with plasmodial myosin partly restored this Ca++-sensitivity. Thus, either myosin or a factor associated with it seems to be involved in the Ca++-dependent regulation of cytoplasmic actomyosin contraction in Physarum. NEM-blocked models reconstituted with skeletal muscle myosin were not reactivated by ATP. The same plasmodia subsequently incubated with plasmodial myosin were able to contract.     

Some Ionic and Bioelectric Properties of the Ameba Chaos chaos

Ionic relationships in the giant ameba Chaos chaos were studied by analyzing bulk preparations of ground cytoplasm for K, Na, and Cl. Ion levels under normal conditions were compared with the levels in cells exposed to varying concentrations of different ions, for varying times and at different temperatures. By standard intracellular electrode techniques, the bioelectric potential, electrical resistance, and rectifying properties of the plasmalemma were studied on intact cells in media of different composition. The results obtained, when related to evidence from other studies on ion fluxes and osmotic relationships, suggest the following concept of ionic regulation in Chaos chaos. In the absence of active membrane uptake, the plasmalemma is essentially impermeable to anions but permeable to both K and Na, which enter passively. In the cold the cell does not discriminate between K and Na, the cytoplasmic level of K + Na is determined by a Donnan distribution, and osmotic imbalance leads to slow swelling. At normal temperatures active processes are added: Na and water are pumped out by the contractile vacuole system; Cl is accumulated, along with the colloid components of the cytoplasm, only during feeding and growth, which depend upon membrane uptake and intracellular membrane transformations. There is no evidence for active transport of any ion species directly across the plasmalemma.     

Oscillating contractions in protoplasmic strands of Physarum: infared reflexion as a non-invasive registration technique

An inexpensive electronic device employing a miniaturised infrared reflection detector is described which enables the automatic registration of intrinsic radial contraction activities of protoplasmic strands of Physarum growing on their original substrate. Advantages and shortcoming of the new registration technique are discussed in relation to recent tensiometric techniques applied to protoplasmic strands for analysing the contraction physiology of cytoplasmic actomyosins, the force generating substrate of the contraction automaticity in the Physarum system.     

Inhibitory Ca(2+)-regulation of myosin light chain kinase in the lower eukaryote, Physarum polycephalum: role of a Ca(2+)-dependent inhibitory factor.

ATP-dependent interactions between myosin and actin in the lower eukaryote, Physarum polycephalum, are inhibited by micromolar levels of Ca2+. This inhibition is mediated by the binding of Ca2+ to myosin, the phosphorylation of which is required if Ca2+ is to inhibit the activities of myosin (Kohama, K., Trends Pharmacol. Sci. 11, 433-435 (1990)). As the first step to examine whether Ca2+ also regulates phosphorylation in the actomyosin system, we purified myosin light chain kinase (MLCK) of 55 kDa almost to homogeneity. The MLCK activity was high whether or not Ca2+ was present. However, a Ca(2+)-dependent inhibitory factor (CIF) purified from Physarum (Okagaki et al., Biochem. Biophys. Res. Commun. 176, 564-570 (1991)) was shown to reduce the MLCK activity in a Ca(2+)-dependent manner. Using crude preparations, not only MLCK but also myosin heavy chain kinase and actin kinase were shown to be inhibited by Ca2+ half-maximally at micromolar levels. Since CIF is the only Ca(2+)-binding protein in the preparations, we propose that this inhibitory Ca(2+)-regulation of the kinases for actomyosin is mediated by CIF.     

等渗NaCL和KCL胁迫对高梁幼苗生长和气体交换的影响

本文比较研究了等渗NaCl和KCl胁迫下,高粱幼苗生长及叶片离子含量、质膜相对透性和有关气体交换参数的变化。结果表明,在低浓度NaCl和KCl胁迫7天时,高粱生长、含水量和质膜相对透性与对照相比没有明显变化,而净光合速率、蒸腾速率和气孔导度已明显下降,叶肉细胞间隙CO2浓度明显增加。NaCl胁迫下叶片Na+含量成倍增加,而K+和Ca2+含量无明显变化。KCl胁迫时叶片K+含量明显增加,Ca2+含量明显下降,而Na+含量没有明显变化。随着NaCl或KCl浓度的增加,幼苗生长和叶片含水量明显下降,质膜透性和细胞间隙CO2浓度明显增加,净光合速率、蒸腾速率和气孔导度进一步下降。 NaCl胁迫下叶片Na+含量进一步增加,K+和Ca2+进一步下降,而KCl胁迫下叶片K+含量进一步 增加,Na+和Ca2+含量进一步下降。KCl对高粱生长抑制、质膜透性、Ca2+含量下降及光合气体交换参数的影响均明显大于等渗的NaCl。     

Reactivation of a cell-free model from Physarum polycephalum: studies on cryosections indicate an inhibitory effect of Ca++ on cytoplasmic actomyosin contraction

Cell-free models should offer "in situ conditions" to study the physiology of cytoplasmic actomyosin in its natural environment, while, if possible, still associated with its regulatory control proteins and other cytoplasmic components. Detergents and glycerol as the usual media to permeabilize the plasmalemma and to extract a portion of the cytoplasmic components, are accompanied by several disadvantages. We investigated a cell-free model consisting of cryosections of plasmodial strands that were previously enriched with "stress fibrils" and fluorescently labelled with phallotoxins and that contain the non-denatured structures that are to be reactivated in situ. The contraction reaction can be directly observed in the fluorescence microscope. This procedure allows the study of contraction conditions in the natural environment of the fibrils. The aim of these reactivation experiments was to identify the role of calcium ions. According to our results, a reactivation of cryosections is not Ca++ dependent but is partly inhibited at concentrations of 10(-4) to 5 X 10(-2)M Ca++. Complete inhibition occurs at 10 to 20 mM Ca++. Electron microscopical investigations revealed that the fluorescently labelled contracting structures were identical to actomyosin fibrils.     

Consequences of impeding in mitochondrial function in Physarum polycephalum. II. Influence on "de novo" generation of contractile activities and responses to glucose and blue light

The "de novo" generation of longitudinal contractile activity in endoplasmic veins is inhibited by 5 mM KCN, whereas 10 mM alpha-ketoglutarate combined with 5 mM AMP abolishes this inhibiting effect in spite of a continued presence of KCN. An analysis of the Young's modulus and studies on the morphogenesis of endoplasmic veins reveal morphological effects of an impediment of cell respiration: (1) an increased fibrillogenesis and changes in the spatial distribution of cytoplasmic actomyosin fibrils, (2) an impediment of the "de novo" generation of the plasmalemma invaginations, and (3) the appearance of a thick cortical layer of ground-plasm. These effects of KCN do not appear in the presence of alpha-ketoglutarate and AMP, and disappear by their subsequent application. Impediment of cell respiration by 5 mM KCN inhibits the tensiometrically registrable responses to glucose and blue light. Both reactions are restored in the presence of KCN by an additional application of 10 mM alpha-ketoglutarate combined with 5 mM AMP. The importance of mitochondrial function with respect to morphogenetic events and to the perception and transduction of external signals as well as to locomotory reactions of Physarum plasmodia is discussed.     

Morphogenesis and Disassembly of the Circular Plasmalemma Invagination System in Physarum polycephalum

During the morphogenesis of small plasmodia developing from endoplasmic drops, an extended plasmalemma invagination system is formed. This system is a characteristic constituent of the ectoplasm. The invaginations have different cytophysiological functions.
The transition from the initial very irregular plasmalemma indentations in protoplasmic drops to the highly organized circular invagination ring of protoplasmic strands, i.e., the differentiation as well as the disassembly of this circular invagination system in retracting endings of strands was investigated with the aid of the semithin- and ultrathin-sectioning technique.
Live observation of protoplasmic drops revealed that simultaneously with the onset of initially irregular oscillating contractions, small endoplasmic streamlets are generated. Subsequently, an improvement of the coordination of contraction activities leads to an oriented mass transport of protoplasm and thereby to locomotion. The growing endoplasmic channels continuously develop into the well-known structure of protoplasmic strands. Differentiation and disassembly of circular plasmalemma invaginations are based on processes of membrane invagination in combination with intracytosis and exocytosis.
The importance and correlations of the following phenomena for morphogenesis and differentiation are discussed: 1) the formation and distribution of the contractile apparatus, i.e., the system of cytoplasmic actomyosin fibrils, 2) plasmalemma invaginations, 3) the generation of oscillating contractions, and 4) the endoplasmic streaming.     

Adenosine triphosphatase distribution in mammary tissue.

Lactating mammary tissue from farm animals and small mammals was perfusion-fixed, prior to histochemical procedures, in an effort to localize the ouabain-sensitive Na+/K+-stimulated ATPase enzyme with the use of specific inhibitors. Histochemical evidence suggests that the Na+/K+-stimulated ATPase is located predominantly on the cytoplasmic side of the plasmalemma in myoepithelial and secretory cells of the alveolus and in epithelial cells of the ducts. Enzyme is not present on the apical plasmalemma of the secretory or duct cells. This substantiates the previous suggestions of localization of the sodium pumps on lateral and basal surface in lactating mammary secretory cells and duct cells.     

The effect of cytoplasmic K+ on the activity of the Na+/K(+)-ATPase.

Experiments with the reconstituted (Na+ + K+)-ATPase show that besides the ATP-dependent cytoplasmic Na(+)-K+ competition for Na+ activation there is a high affinity inhibitory effect of cytoplasmic K+. In contrast to the high affinity K+ inhibition seen with the unsided preparation at a low ATP especially at a low temperature, the high affinity inhibition by cytoplasmic K+ does not disappear when the ATP concentration an-or the temperature is increased. The high affinity inhibition by cytoplasmic K+ is also observed with Cs+, Li+ or K+ as the extracellular cation, but the fractional inhibition is much less pronounced than with Na+ as the extracellular cation. The results suggest that either there are two populations of enzyme, one with the normal ATP dependent cytoplasmic Na(+)-K+ competition, and another which due to the preparative procedure has lost this ATP sensitivity. Or that the normal enzyme has two pathways for the transition from E2-P to E1ATP. One on which the enzyme with the translocated ion binds cytoplasmic K+ with a high affinity but not ATP, and another on which ATP is bound but not K+. A kinetic model which can accommodate this is suggested.     

Cis-allosteric effects of cytoplasmic Na+/K+ discrimination at varying pH. Low-affinity multisite inhibition of cytoplasmic K+ in reconstituted Na+/K(+)-ATPase engaged in uncoupled Na(+)-efflux.

In liposomes with reconstituted shark Na+/K(+)-ATPase the effect of cytoplasmic K+ was investigated in the absence of extracellular alkali ions. During such conditions the Na+/K(+)-ATPase is engaged in the so called uncoupled Na+ efflux mode in which cytoplasmic Na+ activates and binds to the enzyme and becomes translocated without countertransport of K+ as in the physiological Na+/K+ exchange mode. In this uncoupled flux mode only low-affinity inhibition by K+cyt is found to be present. The inhibition pattern is consistent with a model in which cytoplasmic K+ exhibit mixed inhibition of Na+ activation, probably by binding at the three cytoplasmic loading sites on E1ATP (E1A). With determined intrinsic binding constants for cytoplasmic Na+ to this form of KS1, KS2, KS3 = 40 mM, 2 mM, 2 mM the inhibition pattern can be simulated assuming three K+cyt sites with equal affinity for Ki = 40 mM, similar to KS1 for the first Na+cyt site. The discrimination between cytoplasmic Na+ and K+ is therefore enhanced by allosteric interaction initiated from the cis-side due to binding of the first Na+, as opposed to K+, which induces the positive cooperatively in the successive Na+ bindings. pH is found to influence the pattern of K+cyt inhibition: A lowering of the pH potentiates the K+cyt inhibition, whereas at increased pH the inhibition is decreased and transformed into a pure competitive competition.     

Cation selectivity of gastric H,K-ATPase and Na,K-ATPase chimeras.

Chimeras of the catalytic subunits of the gastric H,K-ATPase and Na, K-ATPase were constructed and expressed in LLC-PK1 cells. The chimeras included the following: (i) a control, H85N (the first 85 residues comprising the cytoplasmic N terminus of Na,K-ATPase replaced by the analogous region of H,K-ATPase); (ii) H85N/H356-519N (the N-terminal half of the cytoplasmic M4-M5 loop also replaced); and (iii) H519N (the entire front half replaced). The latter two replacements confer a decrease in apparent affinity for extracellular K+. The 356-519 domain and, to a greater extent, the H519N replacement confer increased apparent selectivity for protons relative to Na+ at cytoplasmic sites as shown by the persistence of K+ influx when the proton concentration is increased and the Na+ concentration decreased. The pH and K+ dependence of ouabain-inhibitable ATPase of membranes derived from the transfected cells indicate that the H519N and, to a lesser extent, the H356-519N substitution decrease the effectiveness of K+ to compete for protons at putative cytoplasmic H+ activation sites. Notable pH-independent behavior of H85N/H356-519N at low Na+ suggests that as pH is decreased, Na+/K+ exchange is replaced largely by (Na+ + H+)/K+ exchange. With H519N, the pH and Na+ dependence of pump and ATPase activities suggest relatively active H+/K+ exchange even at neutral pH. Overall, this study provides evidence for important roles in cation selectivity for both the N-terminal half of the M4-M5 loop and the adjacent transmembrane helice(s).     

The sided action of Na+ on reconstituted shark Na+/K+-ATPase engaged in Na+-Na+ exchange accompanied by ATP hydrolysis. II. Transmembrane allosteric effects on Na+ affinity

The objective of the present investigation was to characterize the ATP-dependent Na+-Na+ exchange, with respect to cation sensitivity on the two aspects of the Na+/K+-pump protein. In order to accomplish this, we used Na+/K+-ATPase reconstituted with known orientation in the proteoliposomes. Activation by cytoplasmic Na+ shows cooperative interaction between three sites. The apparent intrinsic site constants displayed transmembrane dependence on the extracellular Na+ concentration. However, the apparent K0.5 for cytoplasmic Na+ is independent of the extracellular Na+ concentration. The activation by extracellular Na+ at a fixed cytoplasmic Na+ concentration is biphasic with a component which saturates at a concentration of about 1-2 mM extracellular Na+, a plateau phase up to 20 mM, and another component which tends to saturate at about 80 mM followed by a slight deactivation at higher concentrations of Na+. The apparent K0.5 value for extracellular Na+ is also found to be independent of the Na+ concentration on the opposite side of the membrane. The activation by extracellular Na+ can be explained by the negative cooperativity in the binding of extracellular Na+, but positive cooperativity in the rate of dephosphorylation of enzyme species with one and three sodium ions bound extracellularly. Na+ bound to E2-PNa has a transmembrane effect on the cooperativity between binding of cytoplasmic Na+, and E2-PNa2 does not dephosphorylate. K0.5/Vm for cytoplasmic as well as for extracellular Na+ decreases with an increase in the trans Na+ concentration in the non-saturating concentration range. The experiments indicate that at a step in the reaction simultaneous binding of extracellular and cytoplasmic Na+ occurs.     

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.     

Contractile and structural reactions to impediments of Ca2+-homeostasis in Physarum polycephalum

A combined application of 5 mM KCN and 19 microM Ca++-ionophore A-23187 leads to pronounced contractures of plasmodial strands of Physarum polycephalum. The appearance of the contractures is independent of the amount of Ca++ in the external medium. Tensiometric registrations of longitudinal contraction activity (isometric regime) reveal an average tension increase of 50 mp compared with the preceding tension level before the addition of KCN and ionophore. This high force output during the contracture coincides with a pronounced increase in the number of cytoplasmic actomyosin fibrils. Their ultrastructure is seen as a high lateral density of strictly parallel arranged F-actin filaments; the state of cytoplasmic actomyosin during this isometric contracture corresponds to the ultrastructure of isometrically contracted fibrils during the normal contraction-relaxation cycle of this organism. A simultaneous impediment of respiration and Ca++ homeostasis strongly favours a shift of the actin equilibrium to the high polymeric side in the form of fibrils and may thus be used as a preparatory step improving the specimens in the context of other investigations, e.g., for immunocytochemical investigations or for the preparation of cell-free models to be reactivated after extraction procedures.