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.     

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.     

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.     

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.     

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。     

Transmission Electron Microscopy of Meront Development of Eimeria vermiformis Ernst,Chobotar and Hammond, 1971 (Apicomplexa,Eucoccidiorida) in the Mouse,Mus musculus1

First and second generation meronts of Eimeria vermiformis developed in epithelial cells of the crypts of Lieberkühn. They were usually between the host cell nucleus and the basement membrane. Sporozoite organelles dedifferentiated with the first generation meront's development except for the refractile body and the apical complex, which persisted. After several nuclear divisions, the apical complex dedifferentiated further until only micronemes remained attached by a duct system to the plasmalemma. The form of the apical complex was highly variable. Sometimes the duct system was absent and the micronemes were attached directly to the plasmalemma or a dense material on it. Crescent body-like material was often present in the parasitophorous vacuole next to the microneme structure. The microneme structure was not present in second generation meronts but evaginations of the plasmalemma, cytoplasmic outpocketings, and cytoplasmic vesicles were associated with the round granular bodies in the parasitophorous vacuoles. During first generation merogenesis, invaginations from the parasitophorous vacuole formed channels into the meront along which merozoites budded. Micropores were often at the ends of these invaginations. These and other micropores of the meront had a dense U-shaped band for a collar while those of the merozoites had a collar with a double band of dense material that connected to the inner membrane. First generation merozoites budded randomly from the meront, resulting in a residual body that was usually in the middle of the parasitophorous vacuole. Second generation merozoites budded in one direction, resulting in a peripheral residual body and merozoites that were parallel in an oblong parasitophorous vacuole.     

Reactions of yeast cells to glycerol treatment alterations to membrane structure and glycerol uptake

Summary Ultrastructural alterations to the plasmalemma and tonoplast ofSaccharomyces cerevisiae were studied after incubation in hypertonic solutions of glycerol and sorbitol. After 20 to 30 minutes incubation in glycerol, the cells had shrunk to about 40% of their original volume. Large depressions of the plasmalemma were then always found associated with the typical plasmalemma invaginations. The vacuoles of treated cells changed to an irregular form, the tonoplast intramembranous particles were clustered, and large smooth areas appeared. After 6 to 12 hours incubation, cell and vacuole volume, as well as plasmalemma and tonoplast ultrastructure, had reverted to normal. The rate of recovery was strongly temperature dependent.Protoplasts could be similarly shrunk, but no alterations to the plasmalemma ultrastructure were then observed; however, the tonoplast revealed particle clustering as observed in whole cells. Protoplasts also reverted to normal volume and ultrastructure after prolonged incubation. Cells and protoplasts treated with sorbitol showed similar phenomena, but remained shrunken.By the use of radioactive tracers, glycerol was shown to penetrate cells, protoplasts and isolated vacuoles, but no uptake of sorbitol could be demonstrated.During the glycerol permeation period (0.5 to 6 hours), numerous vesicles were found in the cytoplasm and these were possibly engulfed by the vacuole. Associated with the engulfment, patches of tonoplast intramembranous particles were found in a semicrystalline array. Osmotic stress induced alterations to membrane ultrastructure, due to the use of cryoprotective agents, are discussed.A preliminary note of the paper was given at the Sixth European Congress on Electron Microscopy, Jerusalem, 1976.     

Characean charasome-complex and plasmalemma vesicle development

Summary We report on an unusual phenomenon which occurs in some characean algae as a normal plasma membrane activity and also in association with charasome formation. The phenomenon of formation of coated invaginations of the plasma membrane was observed in twoChara and 6Nitella species. These invaginations are coated on their cytoplasmic surface, are 50–60 nm in diameter and rarely exceed 60 nm in length. They are abundant in the young cells ofChara andNitella and also occur in mature cells, but at a lower frequency.N. translucent is an exception in that coated invaginations were few in the young cells and absent in mature cells. Coated vesicles (50–60 nm diameter) were closely associated with these invaginations. Our observations suggest the vesicles may be derived from the invaginations by endocytosis.A close relationship was noted between the development of charasomes (plasmalemma modifications) and coated invaginations. Numerous coated invaginations are seen along the membranes of young charasomes; these invaginations appear to be associated with growth of the charasomes. Coated vesicles were not associated with the coated invaginations of the charasome membrane. The tubular network of cytoplasm and wall space seen in the mature charasome may be formed by fusion of coated invaginations of the developing charasomes, leaving cytoplasmic strands between the fused portions. Coated invaginations were not present along charasomes of the mature cells.     

Endocytosis (heterophagy) in plant cells: Involvement of ER and ER-derived vesicles

The ultrastructure of rice roots water-cultured with hemoglobinwas investigated by electron microscopy. In the cortex cellsof these roots, plasmalemma invaginations were found to be surroundedby ER or ER-derived vesicles. After pinching off from the plasmalemma,invagination-derived vacuoles were surrounded completely byER or ER-derived vesicles. These surrounding structures inflatedand finally joined together to form a new vacuole. This suggeststhat endocytosis involving ER or ER-derived vesicles occursin rice roots. (Received August 24, 1977; )     

Seasonal changes in the ultrastructure of the vascular cambium of Robinia pseudoacacia

 The ultrastructure of the vascular cambium of Robinia pseudoacacia L. was examined in trunk tissues collected over a 2 ¹/₂ year period. During dormancy, fusiform cells are densely cytoplasmic with many small vacuoles and centrally located nuclei. Mitochondria are round to oval in sectional view. The plastids are variable in shape, have few internal membranes, and generally lack starch grains. The plasmalemma is smooth in outline. Proteinaceous material occurs in the vacuoles and many lipid droplets are scattered throughout the ground substance. Smooth tubular ER, often highly dilated, predominates, but short segments of rough ER are also present. Abundant free ribosomes are evenly distributed throughout the ground substance and the dictyosomes are inactive. Microtubules are parietal and have various orientations. During reactivation, the plasmalemma becomes irregular in outline and begins to form invaginations. Concurrently, the proteinaceous material disappears, the vacuoles begin to fuse, polysomes appear, and the dictyosomes begin to produce vesicles. During the period of cambial activity, fusiform cells are highly vacuolate, and the nuclei are centrally located. The mitochondria are round, oval, or elongate. Now the plastids contain phytoferritin, starch grains, or both. Many large invaginations of the plasmalemma intrude into the vacuole, pushing the tonoplast inward and pinching off into the vacuole, which lacks proteinaceous material. Lipid droplets are scarce. Most ER is rough, and ribosomes are generally aggregated as polysomes. Dictyosomes are actively producing vesicles. During the transition to dormancy, the fusiform cells gradually assume the appearance typical of the dormant cambium.     

PINOCYTOSIS IN ROOT CAP CELLS EXPOSED TO URANYL SALTS

In cap cells of intact plant roots exposed to 1mM uranyl for 30 min or less, uranyl crystals were found only in cell walls and in secretory products which had been extruded from the protoplast. In roots exposed for 10–20 hr to 0.1mm uranyl, packets of uranyl crystals bound to secretory products were found within the protoplasts of those exterior cells which contained accumulations of secretory products between the cell wall and protoplast. Although the evidence indicated that these packets of crystals entered the protoplast pinocytotically, results with these specialized exterior cells did not apply to the vast majority of root cap cells in which, after prolonged exposure to 0.1mm uranyl, crystals were concentrated in vacuoles. In roots exposed to 1 or 5mm uranyl for 1 hr, the plasmalemma of interior cap cells was much thicker (13.1 nm) than normal (8.2 nm), and many invaginations and vesicular structures were found near the protoplast surface. Crystals were confined to cell walls except for a few found in vesicles with thickened membranes. Serial sections indicated that most vesicular structures with thickened membranes were in contact with the cell wall, but a few, including some which contained uranyl crystals, were within the protoplast. These results provide evidence of pinocytotic activity in intact plant cells exposed to a toxic heavy metal.     

Discoupling of excitation-contraction processes by urea treatment of frog skeletal muscle

On exposure (E) of frog semitendinosus muscle to 400 mmol/l urea (U) in sodium chloride Ringer's solution, the tension development to isoK+ solutions decreased, while in choline chloride Ringer it increased. On quick removal (R) of urea, always a block of excitation-contraction (E-C) coupling occurred accompanied by transient or persistent swelling of fibres and a similar but definite decrease of their resting membrane potential (Fig. 2). Muscle contraction could be elicited by caffeine even after UER-treatment but then only the slow tension increase (second phase of normal caffeine contraction) occurred (Fig. 3a). The fast tension increase to caffeine (first phase) could be restored if after UER-treatment 5 mmol/l mannitol (Fig. 3b), a 20 min treatment with choline chloride (Fig. 4a) or sodium isethionate (Fig. 4b) Ringer's solution of double osmolarity were applied. Caffeine contraction could not be elicited when sodium chloride Ringer's solution of double osmolarity was used under similar conditions (Fig. 5). E-C block to isoK+ solution persisted in all these experiments. E-C coupling could partially be restored by short treatment of muscle with caffeine (Figs 6a, b).     

STUDIES ON THE ULTRASTRUCTURE OF THE GUARD CELLS OF OPUNTIA

The guard cells of Opuntia contain numerous mitochondria, elements of endoplasmic reticulum, dictyosomes, and microbodies. A complex array of small to large vacuoles which contain small, membrane-bounded vesicles occur in each guard cell. The variety of cytoplasmic constituents and vacuoles suggest that the guard cells are complex in function. A highly reduced grana-fretwork system within the plastids indicates that the photosynthetic capacity of the guard cells is probably rather low. No plasmodesmata occur in the walls between the guard cells and the subsidiary cells while there are numerous invaginations of the guard cell plasmalemmas. Many of the variations in the plasmalemma probably indicate that the plasmalemma is a highly active interface.     

水稻胚乳细胞质膜内陷的超微结构和磷酸酶的细胞化学定位

对生长分化期水稻胚乳细胞的质膜内陷进行了超微结构和磷酸酶的细胞化学研究。结果表明 ,胚乳细胞内的小泡、内质网常与胞间连丝相连 ;质膜形态多变 ,功能活跃 ,由局部起伏的波纹状发展成明显内陷 ,深浅不一 ,多呈袋状 ,袋中包含着大小不一的泡状物 ;有些内陷脱离质膜成为胞质中的囊泡 ,表现出活跃的内吞现象。除细胞间隙中含有圆球状的内含物外 ,在质膜内陷和囊泡中常含有大量的内含物。H ATP酶定位结果显示 ,质膜及其邻近的泡状物周围有酶的分布 ;而酸性磷酸酶定位在液泡、胞间隙和其中的泡状内含物周围 ;在质膜及其内陷形成的囊泡中有G6P酶的分布。这些结果表明胞间隙和质膜内陷在物质的运输中可能起着重要作用     

Invaginations in plant cell protoplasts containing mycoplasma-like bodies (MLO)

MLO containing invaginations were found in protoplasts of phloem parenchyma cells in symptomless young leaves ofRibes houghtonianum Jancz. infected with a yellows disease. The invaginations originate between the cell wall and plasmalemma, usually at plasmodesmata, and change apparently into superficial vesicles in the protoplast; they are entirely or partially limited by host plasmalemma. The formations mentioned occur in parenchyma cells which contain normal organelles. Sometimes they are divided by a smooth membrane system enclosing MLO. Besides MLO the invaginations contain in some cases slimy fibrils resembling the P-protein in sieve tubes. The MLO bodies seen in invaginations have usually a diameter of 50–250 nm and their plasmalemma (unit membrane) is identical with the plasmalemma of MLO bodies occurring in sieve tubes. However, only few MLO bodies in invaginations are electron dense, so that they resemble naturally degenerated forms of MLO. Similar MLO containing invaginations were formerly described from some leafhoppers transmitting MLO.     

Rapid, extensive and reversible vacuolation of Schizosaccharomyces pombe induced by amphotericin B

Abstract The fission yeast Schizosaccharomyces pombe has no large vacuoles under normal growth conditions, although budding yeasts usually have large central vacuoles. The minimum inhibitory concentration of amphotericin B to S. pombe was 0.5 μg ml⁻¹; treatment with 0.2 μg ml⁻¹ for 20 min induced rapid and extensive vacuolation in S. pombe exponential phase cells. Growth rate of the cells with 0.2 μg ml⁻¹ amphotericin B was much reduced for 6 h, showing extensive vacuolation. Vacuolation in itself was not fatal: on removal of the drug, most cells recovered gradually and eventually multiplied.     

Binding and uptake of 125I-insulin into rat liver hepatocytes and endothelium. An in vivo radioautographic study

Electron microscope radioautography has been used to study hormone-receptor interaction. At intervals of 3, 10, and 20 min after the injection of 125I-insulin, free hormone was separated from bound hormone by whole body perfusion with modified Ringer's solution. The localization of bound hormone, fixed in situ by perfusion with glutaraldehyde, was determined. At 3 min, 125I-insulin has been shown to be exclusively localized to the hepatocyte plasmalemma (Bergeron et al., 1977, Proc. Natl. Acad. Sci. U. S. A., 74:5051--5055). In the present study, quantitation indicated that 10(5) receptors were present per cell and distributed equally along the sinusoidal and lateral segments of the hepatocyte plasmalemma. At later times, label was found in the Golgi region. At 10 min, both secretory elements of the Golgi apparatus and lysosome-like vacuoles were labeled, and at 20 min the label was especially concentrated over the latter vacuoles. Acid phosphatase cytochemistry showed that the vacuoles did not react and therefore were presumed not to be lysosomal. These Golgi vacuoles may constitute a compartment involved in the initial degradation and/or site of action of the hormone. Control experiments were carried out at all time intervals and consisted of parallel injections of radiolabeled insulin with excess unlabeled hormone. At all times in controls, label was diminished over hepatocytes and was found primarily over endothelial cells and within the macropinocytotic vesicles and dense bodies of these cells. Kupffer cells and lipocytes were unlabeled after the injection of 125I-insulin with or without excess unlabeled insulin.     

Pinocytosis in the root cells of a salt-accumulating halophyte <Emphasis Type="Italic">Suaeda altissima</Emphasis> and its possible involvement in chloride transport

Ultrastructure of root cells in salt-accumulating halophyte Suaeda altissima (L.) Pall. was examined with transmission electron microscopy. Plants were grown hydroponically on nutrient media containing 3, 50, 250, and 500 mM NaCl. Some plants were exposed to hypersomotic salt shock by an abrupt increase in NaCl concentration from 50 to 400 mM. Growing S. altissima plants at high NaCl concentrations induced the formation of type 1 pinocytotic structures in root cells. Type 1 structures appeared as pinocytotic invaginations of two membranes, the plasmalemma and tonoplast. These invaginations into vacuoles gave rise to freely ‘floating’ multivesicular bodies (MVB) enclosed by a double membrane layer. The pinocytotic invaginations and MVB contained the plasmalemma-derived vesicles and membranes of endosome origin. The hyperosmotic salt shock led to formation of type 2 and type 3 pinocytotic structures. The type 2 structures were formed as pinocytotic invaginations of the tonoplast and gave rise to MVB in vacuoles. Unlike type 1 MVB, the type 2 MVB had only one enclosing membrane, the tonoplast. The type 3 structures appeared as the plasmalemma-derived vesicles located in the periplasmic space. The cytochemical electron-microscopy method was applied to determine the intracellular Cl^? localization. This method, based on sedimentation of electron-dense AgCl granules in tissues treated with silver nitrate, showed that the pinocytotic structures of all types contain Cl^? ions. The presence of Cl^? in pinocytotic structures implies the involvement of these structures in Cl^? transport between the apoplast, cytoplasm, and the vacuole.     

Ultrastructural Studies on the Secretory Cavity Development and Essential Oil Accumulation in the Fruits of Evodia rutaecarpa

The secretory cavity in fruits of Evodia rutaecarpa (Juss.) Benth. developed schizogenously through a separation of the walls of the central initial cells. Electron micrographs revealed that in the early stages of cavity development there was an apparent increase in the number and volume of the plastids in which esmiophilic droplets and tubular dements were observed. This suggested that the essential oils might be synthesized in the plastids.. The essential oils were then transport ed through the plastid membrane to the surrounding endoplasmic reticulum or into the vacuoles, becoming vesicles approaching the plasmalemma, and finally releasing their contents into the oil chamber by plasmalemma invaginations.     

Oscillating contractions in protoplasmic strands of Physarum: effects of external Ca++-depletion and Ca++-antagonistic drugs on intrinsic contraction automaticity

1. The minimal requirement of external Ca-concentration for continuance of contraction activity lies in the range of 10(-4) M. 2. As in mammalian smooth muscle, the Ca antagonistic drugs verapamil and D 600 (5.10(-4) M) suppress the minute-rhythms. The action of the drugs is inhibited by 5mM Ca, La or Mn. De novo generation of contraction automaticity is not inhibited by external Ca depletion or by Ca antagonists. 3. It is concluded that rhythmical Ca fluxes across the cortical plasmalemma are not a precondition for triggering continuance or de novo generation of contraction automaticity.