Plasmalemma invaginations, contraction and locomotion in normal and caffeine-treated protoplasmic drops of Physarum

The de novo formation of plasmalemma invaginations and vacuoles of light microscopic dimension in protoplasmic drops at different age-stages was studied quantitatively by applying morphometric methods and marker techniques. The study includes: i) the normal morphogenetic development to a drop age of 1 hour, ii) the influence of caffeine treatment, and iii) the effects of removal of this drug. In untreated drops, formation of invaginations and vacuoles is accomplished within 10 to 15 min. By application of 5 mM caffeine, the formation of plasmalemma invaginations is inhibited for 20 to 30 min. The onset of oscillating contraction activity is delayed, but not hindered by the drug. Drug removal 20 min after drop generation leads to an immediate initiation of plasmalemma infolding. Although caffeine does not hinder initiation of normal contraction activity, the locomotory ability of the drop is blocked if the drug is not removed from the drop and the substrate. Thus, caffeine uncouples motive force generation from locomotion in all plasmodial stages of Physarum. The cellular sites of drug action are 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.     

Immunocytochemistry of the acellular slime mold Physarum polycephalum

Abstract. The polygonal arrangement of actomyosin fibrils in different stages of the acellular slime mold Physarum polycephalum is correlated with morphogenetic processes at the cell surface. Light and electron microscopic investigations on both endoplasmic drops and thin-spread small plasmodia demonstrate that the differentiation of a polygonal pattern depends on a transient deficiency of plasma membrane invaginations.
Glycerol-extracted specimens show condensation and drastic spatial changes in the organization of the polygonal net after addition of ATP, thus indicating contractile properties of this system. Observations with the polarizing microscope reveal rhythmic changes in fibrillar birefringence intensity corresponding to the protoplasmic streaming activity, i.e., birefringence increases during contraction and decreases during relaxation. Cell fusion experiments, local irradiation with blue light (450 nm), and chemical treatment by impeding the mitochondria1 function with DNP (2,4-di-nitrophenol) demonstrate morphological as well as physiological interdependences of the actomyosin system, the motive force generation, and the expression of a locomotor polarity in plasmodia of Physarum polycephalum.     

Effects of caffeine and D2O on persistence and de novo generation of intrinsic oscillatory contraction automaticity in Physarum

The present investigation was performed in an attempt to contribute to answering the question whether the plasmalemma of the plasmodial stage of Physarum represents the site of a trigger mechanism for the oscillating contraction activity of cytoplasmic actomyosin. The effects of the following substances on persistence of tensiometrically measured longitudinal and radial activities of Physarum veins and on de novo generation of activities in experimentally generated drops were studied: caffeine, theophylline, acetylcholinium chloride, procaine, physostigminium salicylate, iso-ompa, nifedipin, sodium nitroprusside, potassium thiocyanate, D2O; as well as the effects of ions such as La+++ and high outer concentrations of Na+ and K+. Some of the substances were applied simultaneously for comparison externally (by bathing solutions) and internally (by injection). The experimental data speak against the existence of electrogenic rhythmical Ca++, Na+ or K+ pumps across the plasmalemma which could have a triggering function for the oscillation. The contraction activities of the cytoplasmic actomyosin seem to represent a spontaneous endogeneous oscillation which can be modulated via the plasmalemma during chemotaxis.     

Progress in plasmodial differentiation improves regularity of oscillating contractions in Physarum polycephalum

Based on the knowledge about subcellular morphogenetic processes in the acellular slime mold Physarum polycephalum, we hypothesized that during differentiation of undifferentiated endoplasm to the highly differentiated complex structure of the contractile apparatus of this organism, the regularity of oscillating contractions must improve. We measured the endogenous contraction automaticity starting from the de novo generation within minutes after sampling small portions of undifferentiated endoplasm. The standard deviation of the normalized period duration of these samples was compared to the respective values of radial contractions of differentiated protoplasmic plasmodial strands. The mean normalized standard deviation in endoplasmic drops was 28.3+/-12.2%. Respective values in protoplasmic strands were 10.0+/-3.7%. The difference between the experimental groups was highly significant (p<0.0001). We interpret the verification of our hypothesis as an indication that the very regular oscillating contractions in fully differentiated stages of Physarum require the complex structure of the sophisticated contractile apparatus, represented by the circular plasmalemma invagination system of protoplasmic strands, while the regularity is lower in stages, where the differentiation is still in progress. We believe that this is due to deficits in coordination capabilities, which need a directional and spatially oriented protoplasmic streaming as a precondition.     

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.     

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.     

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.     

Transformation of cytoplasmic actin importance for the organization of the contractile gel reticulnm and the contraction — relaxation cycle of cytoplasmic actomyosin

(1) Within the low viscous flowing endoplasm of Physarum polycephalum a considerable amount of actin is in the non-filamentous state. This can be demonstrated by applying poly-L-lysin to surface spreads of native protoplasm. (2) It has been shown that in protoplasmic drops the endoplasm-ectoplasm transformation is accompanied by an actin polymerization from the non-filamentous state to F-actin. (3) The actual state of the labile G-F-actin equilibrium determines the varying consistency (viscosity) of the cytoplasm. (4) Increasing viscosity can be interpreted as being brought about by a) shifting of the G-F-actin equilibrium to the filamentous side, and (b) increased myosin-mediated binding sites between actin filaments. (5) Polymerization and depolymerization processes are involved in the rhythmically occurring contraction-relaxation cycle of cytoplasmic actomyosin in Physarum. (6) Cytoplasmic actin and myosin represent the architectural proteins of the contractile gel reticulum in eukaryotic cells. (7) The importance of the regulation of actin polymerization as a basic control mechanism of the eukaryotic cell is discussed.     

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.     

甜菊愈伤组织中的质膜内陷:超微结构和酸性磷酸酶细胞化学定位

对在分化条件下的甜菊 (Stevia rebaudiana)愈伤组织分生区域细胞的质膜内陷进行了超微结构和酸性磷酸酶细胞化学研究。结果表明 ,在不同液泡化状态的细胞中均有质膜内陷存在。在原生质浓密的细胞中 ,质膜呈起伏的波纹状 ,某些部位发生明显内陷 ,大小不等 ,多呈圆球状。在部分液泡化细胞中 ,质膜内陷体积增大 ,内含物增多且结构复杂。在液泡化细胞中 ,质膜内陷嵌入中央液泡 ,但彼此间以一膜间隙隔开。质膜内陷中的内含物以小泡和卷绕的膜结构形式存在。酸性磷酸酶活性定位结果显示 ,质膜及其内陷含高的酶活性。推测质膜内陷在功能上与液泡相似 ,构成了这些细胞水解空间的一部分。     

Cycling aggregation patterns of cytoplasmic F-actin coordinated with oscillating tension force generation

Summary Isometric contracting protoplasmic veins of Physarum polycephalum show cycling patterns of cytoplasmic F-actin, dependent on their oscillating contraction behaviour (minute rhythms). The process of fibrillogenesis represents a parallel arrangement of F-actin chains (plasma filaments, microfilaments) during the isometric contraction phase. A part of the results of the present work corroborates previous results on stretch-activated veins which showed that the fibrillar form of F-actin reflects the isometric contracted state.During isometric relaxation phase, a disaggregation of the fibrillar pattern takes place and is accompanied by a deparallelisation of F-actin chains. Therefore, the isometric relaxed state of cytoplasmic actomyosin is non-fibrillar in nature. Thus, the morphologically detectable fibrillar form of cytoplasmic actomyosin, according to physiological interpretation, is solely representative of the isometric contracted state.The question whether assembly-disassembly processes, e.g. GF-actin-transformation, play a role in the contraction-relaxation cycle is discussed.Dedicated to Prof. Dr. Dr. h.c. W. Bargmann on the occasion of his 70 birthday. — Paper presented at the Cell Surface Project Group workshop Interrelation of cell surface and cell locomotion, European Organization for Research on Treatment of Cancer (EORTC), June 6–8, 1975, Zürich, Switzerland.     

Plasmalemma Invaginations in Cultured Callus of Stevia rebaudiana: Ultrastructure and Ultracytochemical Localization of Acid Phosphatase

The plasmalemma of cells within meristematic regions was observed to possess invaginations in cultured callus of Stevia rebaudiana under differentiation. The ultrastructure and acid phosphatase (AcPase) ultracytochemistry Of these invaginations were studied. The plasmalemma invaginations occurred in the cells at various stages of vacuolation. In cells with dense protoplasm, plasmalemma appeared undulated but occasionally spherical and variable in size with conspicuous invaginations that projected into the peripheral cytoplasm. In the partially vacuolated cells, plasmalemma invagination became voluminously enlarged with increased contents and structurally complexed. In vacuolated cells, the enlarged invaginations protruded into the central vacuole but were delimitted from the tonoplast by an intermembrane zone continuous with the peripheral cytoplasm. Complex accumulations of membranes consisting of vesicular and coiled membranous Structures might develop within the plasmalemma invaginations. AcPase localization demonstrated high enzymic activity in the plasmalemma and its associated invagination. It seemed likely that these invaginations were functionally analogous to the vacuoles and therefore constituted part of the lytic compartment in these cells.     

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.     

Freeze-fracture studies of the plasmalemma of Candida albicans after treatment with econazole-nitrate.

In electron microscopic studies the interior of the plasmalemma of Candida albicans was revealed by means of the freeze-fracture technique. The superficial structures of the extracellular (E) and protoplasmic (P) fracture faces differed negligibly from structures on the corresponding fracture faces of Saccharomyces cerevisiae. Following treatment with 2.2 x 10(-5) M econazole nitrate a layer, present on the P face in the form of a tight matrix of globular proteins, dissolved into isolated groups of particles whose globular elements sometimes formed hexagonal patterns. As the damage progressed, fissure-shaped membrane invaginations on the P face disappeared. Parts of the outer lipid layer of the plasmalemma were torn off the cell wall and adhered in fragments to the P face. The ultrastructural changes in the plasmalemma induced by econazole nitrate temporally correlate with an increase in the permeability of the cell envelope found in physiological studies performed by other authors.     

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.     

Studies on microplasmodia ofPhysarum polycephalum

Summary The new technique of fluorescent analog cytochemistry was used to investigate the cell surface morphology (RITC-WGA staining), the organization of the microfilament system (Rh-phalloidin staining) and the spatial distribution of mitochondria (Rh-123 staining) in the various growth stages of axenically cultured living and fixed microplasmodia ofPhysarum polycephalum. The differentiation degree of the cell surface is generally size- and age-dependent: the invagination system develops by degrees from small spherical stages (50–100 m) without invaginations to large vein-like or dumbbell-shaped specimens (300–1,000 (m long) with extensive invagination systems. The microfilaments are always organized in a cortical system along the entire cell surface and sometimes in a fibrillar system as well, extending throughout the cytoplasmic matrix. Results on living microplasmodia demonstrate that the cortical microfilament system is mainly involved in motive force generation for changes of cell surface morphology and protoplasmic streaming activity, whereas the fibrillar system rather serves a stabilizing and adhering function. Moreover, the functional differences of the two microfilament systems are indicated by the position of a large population of stationary mitochondria in close vicinity to the cell surface, thus pointing to a reasonable arrangement of the energy-supplying and energy-transforming system.     

Die Regeneration des Plasmalemms vonPhysarum polycephalum

Zusammenfassung Die Regeneration des Plasmalemms vonPhysarum polycephalum erfolgt nach experimenteller Ruptur sowohlbei Protoplasmatropfen als auch bei isoliertem Protoplasma durch Membranvesikulationsprozesse. Verantwortlich für die Neubildung des Plasmalemms sind verschiedene protoplasmatische Vakuolen (Schleimvakuolen, Nahrungsvakuolen). Unmittelbar nach experimenteller Entfernung des Plasmalemms kommt es zu einer Ansammlung von Vakuolen in einer 5–10 tiefen Region an der Oberfläche des nackten Protoplasten. Durch successive Konfluation dieser Vesikel entstehen zunächst einige gro\e, parallel zur Protoplasmaoberfläche abgeflachte Vakuolen, die schlie\lich zu einer einzigen, das gesamte Protoplasma allseitig umschlie\enden Vakuole verschmelzen. Diedistale Vakuolenmembran zerfällt in einzelne, unterschiedlich gro\e Areale, die zusammen mit degenerierenden Bestandteilen des Protoplasmas abgesto\en werden. Dieproximale Vakuolenmembran wird zur neuen Oberfläche des überlebendenPhysarum-Protoplasmas. Der gesamte Vorgang der Membran-Neubildung erfolgt innerhalb von 5–6 sec und scheint unabhängig von der Natur des umgebenden Milieus zu verlaufen. Trotz der Tatsache, da\ innerhalb der ersten Phase des Regenerationsprozesses das Protoplasma kurzfristig nackt war, d.h. ohne membranöse Begrenzung existierte, lä\t sich keine Einwanderung von partikulären Substanzen des Au\enmediums in das überlebende Protoplasma nachweisen, wie durch Versuche mit dem MarkierungsmittelAerosil gezeigt werden konnte.

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The regeneration of the plasmalemma of Physarum polycephalum

Summary After experimental rupture, the plasmalemma of protoplasmic droplets and isolated protoplasm ofPhysarum polycephalum regenerates by means of membrane vesiculation. Various (mucus- and food-) vacuoles are responsible for the new formation of the plasmalemma. Immediately after removal of the old plasma membrane, many vacuoles accumulate below the boundary between the protoplasm and the surrounding medium in a region of 5–10 depth. Later some large vacuoles, which are strongly flattened parallel to the surface of the protoplasm, originate by means of successive confluence of these single vesicles. The large vacuoles finally fuse to a single vacuole which surrounds the whole protoplast. Thedistal membrane of this vacuole disintegrates together with degenerating protoplasmic components. Theproximal membrane of the vacuole represents the new surface of the surviving protoplasm. The process of regeneration of the plasmalemma takes 5–6 sec and seems not to depend on the kind of medium surrounding the protoplasm. Inspite of the fact that during a part of the first phase of regeneration, the cytoplasm is naked (i.e. without a membranous boundary), there is no migration of particulate substances from the outer medium into the surviving protoplasm, as shown by experiments with the marker substanceaerosil.

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Frau B. Koeppen, Fräulein I. Kletschke und Frau M. Sauernheimer danken wir für technische Hilfe.     

Extraction of an actin-like protein from the plasmodium of a myxomycete and its interaction with myosin A from rabbit striated muscle

An actin-like protein was obtained from the plasmodia of a myxomycete, Physarum polycephalum. It forms a complex with muscle myosin A which behaves similarly to the actomyosin from rabbit striated muscle. On the addition of ATP the complex of this protein with myosin A shows a viscosity drop at high concentrations of KCl (～0.5 M). At low concentrations of KCl (～0.05 M) this complex superprecipitates from solutions containing 1 mM MgCl₂ and shows Mg-activated ATPase activity. That is, the actin-like protein converts the ATPase of myosin A to the actomyosin type.     

Plasmalemma structure in freezing tolerant unicellular algae

Summary Electron microscopy of several freezing tolerant species of the algal generaChlamydomonas andChloromonas revealed plasmalemma invaginations which are absent from freezing sensitiveChlamydomonas species. The morphology of these invaginations in freezing tolerant strains is described and compared with similar structures in the yeastSaccharomyces cerevisiae.