Morphological evidence for the existence of a more complex cytoskeleton in Amoeba proteus

Summary Various stabilization and extraction procedures were tested to demonstrate the ultrastructural organization of the cytoskeleton in normal, locomoting Amoeba proteus. Most reliable results were obtained after careful fixation in glutaraldehyde/lysine followed by prolonged extraction in a polyethylene glycol/Triton X-100 solution. Before dehydration in a graded series of ethanol and critical-point drying, the amoebae were split by the sandwich-technique, i.e., by mechanical cleavage of cells mounted between two poly-L-lysine-coated glass slides. Platinum-carbon replicas as well as thin sections prepared from such cell fragments revealed a cytoskeleton composed of at least four different types of filaments: (1) 5–7-nm filaments organized as a more or less ordered cortical network at the internal face of the plasma membrane and probably representing F-actin; (2) 10–12-nm filaments running separately or slightly aggregated through the cytoplasm and probably representing intermediate filaments; (3) 24–26-nm filaments forming a loose network and probably representing microtubules; and (4) 2–4-nm filaments as connecting elements between the other cytoskeleton constituents. Whereas microfilaments are responsible for protoplasmic streaming and other motile phenomena, the function of intermediate filaments and cytoplasmic microtubules in amoebae is still obscure.     

Dynamics of the cytoskeleton in Amoeba proteus

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.     

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.     

Dynamics of the cytoskeleton inAmoeba proteus: II. Influence of different agents on the spatial organization of microinjected fluorescein-labeled actin

Summary Iodoacetamido-fluorescein-(IAF)-labeled actin was microinjected into normal locomotingAmoeba proteus. Thereafter (30–60 minutes) changes in the cytoplasmic fluorescence distribution pattern and contractile activity were induced by internal and external chemical stimulation. Different agents such as phalloidin, procaine, 2.4-dinitrophenol (DNP), puromycin, ouabain and n-ethyl maleimide (NEM) interfere with the excitation-contraction mechanism involved in ordered pseudopodium formation during ameboid movement and cause various morphogenetic reactions based on actin polymerization-depolymerization cycles. Most frequent changes are (a) local condensation of IAF-actin and formation of a continuous IAF-actin layer at the cytoplasmic surface of the cell membrane and around the pulsating vacuole, (b) immobilization and hyalo-granuloplasm separation by combined contraction and detachment of the IAF-actin layer from the cell membrane, (c) organized and disorganized formation of pseudopodia by local contraction and disintegration of the IAF-actin layer, and (d) alterations in the rheological properties of the protoplasmic matrix by changes in the molecular state of soluble actin not incorporated into the cytoskeleton. The experimental approaches to the function of the actomyosin system in large amebas attainable by the method ofin vivo molecular cytochemistry are discussed in detail with respect to the participation of the cytoskeleton in motive force generation for cytoplasmic streaming and ameboid movement.     

Effects of induced pinocytotic activity and extreme temperatures on the morphology of golgi bodies in Amoeba proteus

Summary 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° C and 30° 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° C and 38% at 30° 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 3 h, 4 h 10 h and 15 h at 4° C and 30° 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.     

Pinocytose und Bewegung von Amöben

Zusammenfassung Zur Bestimmung des cytotischen Membran-Turnovers wurden morphometrische Messungen an über 60 Zellen der Art Amoeba proteus durchgeführt. Danach nehmen diese Amöben 0,14% ihrer Zelloberfläche pro Minute durch permanente Endocytose in das Cytoplasma auf. A. proteus benötigt also insgesamt 12 Std, um die gesamte Zellmembran während der normalen Bewegung einmal zu erneuern. Infolge des geringen Membranturnovers kann der permanenten Endocytose keine aktive Bedeutung für die Erzeugung der Bewegungstriebkraft zugesprochen werden. In Übereinstimmung mit dieser Vermutung ließ sich eine Abhängigkeit zwischen Fortbewegungsgeschwindigkeit und Endocytoseintensität nicht nachweisen.Entsprechende Messungen mit drei verschiedenen Endocytoseinduktoren ergaben für die induzierte Endocytose in Abhängigkeit von der verwendeten Substanz eine wesentlich höhere Ingestionsrate von 0,43–2,25%/min. Derartige Spitzenwerte können allerdings nur innerhalb eng begrenzter Zeiträume von 15–30 min erzielt werden. Vergleicht man dagegen die Membranaufnahme während der permanenten und induzierten Endocytose über längere Zeitintervalle (4–5 Std), so bleibt die induzierte Endocytose mit 0,05–0,12%/min in der Intensität deutlich hinter der permanenten Endocytose (0,14%/min) zurück. Eine Erhöhung der Temperatur auf 30° und eine Erniedrigung auf 15°C bringen beide Endocytoseformen zum Erliegen.Die permanente Endocytose muß bei Amöben neben der Phagocytose als der wichtigste Mechanismus zur kontinuierlichen Aufnahme gelöster und suspendierter Stoffe (bis zur Größenordnung von Bakterien) angesehen werden.

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Pinocytosis and locomotion of amoebae

Summary Cytotic membrane turnover of Amoeba proteus was morphometrically studied in more than 60 cells. The results obtained indicate that 0.14% of the total cell membrane area per minute is ingested by permanent endocytosis. Consequently during normal locomotion the total cell membrane area is renewed once within 12 hours.This rate is too low to play any role in the generation of motive force. No correlations were found between the rates of locomotion and permanent endocytosis.Comparative measurements on cells treated with three different substances inducing endocytosis reveal that induced endocytosis leads to an increased rate of membrane ingestion of 0.43–2.25%/min depending on the substance used. These high rates, however, are only maintained during short periods of time (15–30 min). When the rates are calculated on the basis of long periods of time (4–5 hours), it is obvious that induced endocytosis (0.05–0.12%/min) is less effective in long term membrane turnover than permanent endocytosis (0.14%/min). Endocytotic activity is completely abolished by both the increase and decrease in temperature to 30°C and 15°C respectively.In addition to discontinuous phagocytosis permanent endocytosis is an important mechanism for continuous ingestion of fluid including particles up to the size of bacteria.

Der Kultusminister des Landes Nordrhein-Westfalen unterstützte die Untersuchung aus Überschußmitteln des Westdeutschen Rundfunks.     

Intracellular pathways and degradation of endosomal contents in basal epithelial cells of freshwater sponges (Porifera, Spongillidae)

 The digestive system expressed by basal epithelial cells of the freshwater sponges Spongilla lacustris and Ephydatia muelleri is mainly represented by a population of 30–50 preexisting lysosomes located in the close vicinity of the central nucleus. The strongly acidic vacuoles (pH 4–4.5) vary in size between 1 and 3 μm, and contain a set of different lysosomal enzymes. Immunocytochemical studies succeeded in the detection of β-hexosaminidase, cathepsin D, acid phosphatase, and α-glucosidase. Endosomes resulting from fluid-phase macropinocytosis, receptor-mediated endocytosis, or phagocytotic activity deliver their exogenous contents to the preexisting lysosomes for enzymatic degradation. Macropinosomes and phagosomes follow a rather reduced intracellular pathway by immediate fusion with the lysosomal compartment, whereas substances conveyed by coated vesicles pass through two additional vacuolar stages, namely early and late endosomes. Early endosomes serve as sorting organelles and segregate various constituents of complex ligands (BSA-AU_6, BSA-AU₁₂) by size into individual late endosomes, which then coalesce with preexisting lysosomes. As a whole, the intracellular pathways and hydrolytic processing of endosomal and phagosomal contents in freshwater sponge cells share certain similarities with the respective mechanisms in cells of higher eukaryotes. Accepted: 7 October 1997