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.     

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.     

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.     

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.     

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。     

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.     

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     

银耳（Tremella fuciformis）原基分化前期双核菌丝细胞和分生孢子的边缘体与质膜体

本文报道银耳（Ｔｒｅｍｅｌｌａｆｕｃｉｆｏｒｍｉｓ）原基分化前期．在双核菌丝的幼细胞、成熟细胞和分生孢子中,与质膜相关联的两类膜结构──边缘体和质膜体的形成与功能。根据相似结构的存在．支持小泡或多泡体排出质膜之外附在细胞壁上成为边缘体和参于细胞壁合成的假定。银耳原基分化前期．双核菌丝迅速分裂的幼细胞．其质膜内陷产生泡状质膜体,内含数个小泡,或产生膜状质膜体;在成熟细胞中．质膜内陷通常形成回旋的膜结构──膜状质膜体．内含１—２个电子致密小泡．当这两类质膜体脱离质膜进入细胞质后,有的膜层和小泡局部被消化．因此,推断质膜体具有内吞和输送养料的作用。另外．在桶孔隔膜闭塞一侧电子致密度高的细胞质中．还观察到一种罕见的只有单个膜层的质膜体．其内充满３个电子致密小泡．估计它的形成与功能同膜状质膜体相似。作者认为．桶孔闭塞和质膜体的出现是与银耳原基细胞分化有关联的两个重要特征。最后,在成熟细胞中,尚可以观察到质膜体的膜层能够散开形成内质网．因此．内质网也可以来源于质膜体。     

Differentiation and Ultrastructure of the Protophloem Sieve Elements of Minor Veins in the Maize Leaves: Changes in the Protoplast

Protophloem sieve element differentiation in the minor veins of the maize ( Zea mays L. ) leaves was first evidenced as an increase of the wall thickness, which began in the comers of the cell and then extended to other parts of the wall, and the appearance of long rough endoplasmic reticulum cistemae distributed throughout the cytoplasm, and then the presence of characteristic crystalloid inclusions within the plastids. As differentiation progressed, long cisternae of rough endoplasmic reticulum appeared to transform into shorter forms and eventually aggregated into small stacks, losing their ribosomes during the process. The nuclei degenerated, although frequently persisted until very late in differentiation the stages of maturation, as darkly stained amorphous aggregates surrounded by double nuclear envelope or only inner membrane of nuclear envelope. Subsequently, the nuclear envelope collapsed and became discontinuous. At the beginning of nuclear degeneration the perinuclear spaces were partly dilated and sometimes the outer nuclear envelope in the dilated portions then ruptured, and was accompanied by the disappearance of the cytoplasmic portion near it. During the peried of nuclear degeneration, in addition to the endoplasmic reticulum, plastids and mitochondria underwent structural modification, while components such as ribosomes, cytoplasmic ground substances, vacuoles and dictyosomes disintegrated and disappeared. At maturity, the surviving protoplasmic components, including plasmalemma, mitochondria, small stacked smooth endoplasmic reticulum and P-type plastids with crystalloids, became parietal in position. As differentiation of adjacent metaphloem sieve elements proceeded, the protoplasmic components of the mature protophloem sieve elements progresively degenerated and finally obliterated.     

Lomasomes and plasmalemmasomes in fungi

Summary Electron microscope observations of fungal hyphae and yeast-like cells, using conventional fixation methods and freeze-etching, demonstrate that plasmalemmasomes are not fixation artifacts. The small invaginations of the plasmalemma observed in aldehyde-fixed preparations are also present in frozen-etched samples. The morphology of plasmalemmasomes in the species examined is variable and ranges from vesicles or tubules within the plasmalemma invagination to parallel arrays of membrane lamellae. Plasmalemmasomes thus appear to be primarily excess plasma membrane that has accumulated, perforce, endocellularly. Lomasomes, in contrast, appear to be accumulations of ejected material between the plasmalemma and cell wall that have become sequestered by the deposition of wall material.     

A Translocation Hypothesis based on the Structure of Plant Cytoplasm

Two types of linear structures have been seen in plant cytoplasm,largely by phase-contrast microscopy. Microscopic fine threads,o.Iµ to Iµ in diameter, were revealed in hair cells,where they formed endoplasmic systems along streaming pathwaysin the parietal cytoplasm and in transvacuolar strands. Duringcirculation streaming, small plastids and mitochondria-likeparticles were observed moving along the fine threads. Similarfine threads, together with small plastids and mitochondria-likeparticles occurred in phloem exudate and transcellular microscopicstrands. The transcellular strands, Iµ to 7µ indiameter, were seen in sieve-tube elements, phloem parenchyma,border parenchyma, and cortical cells. The movement of small plastids across end walls in border-parenchymacells, the appearance of the same structures within strandsin phloem cells, and the undiminished occurrence of small plastidsin successive drops of phloem exudate are collectively takenas evidence for their participation in translocation. Particlemovement is thought to occur through transcellular strands inassociation with fine threads, and to be motivated by a transcellularform of protoplasmic streaming.     

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.     

Morphology ofEntomophthora muscae protoplasts grownin vitro

Summary Entomophthora muscae (C.) Fres. can be grownin vitro as protoplasts. Light and electron microscopical studies of thein vitro developed protoplasts have demonstrated the absence of an organized wall over the protoplasmic Con A-positive membrane at all stages of growth. The cytological organization is typical of the Entomophthorales with condensed chromatin in the interphase nuclei and small eccentric metaphase spindles. Long strands of endoplasmic reticulum, microubules and vesicles surrounding the plasmalemma may be involved in maintaining the precise shape ofE. muscae protoplast. Starvation of the fungus induces the formation of hyphal bodies after deposition of Con A- and WGA-positive wall material at the plasmalemma surface.Abbreviations Con A concanavalin A - DH Drosophila cell culture medium - FITC fluorescein isothiocyanate - GLEN glucose-lactal-bumin-yeast extract-NaCl culture medium for protoplasts - HBL hyphal body-like protoplasts - MM Mitsuhashi and Maramorosch' insect cell culture medium - PATAg periodic acid-thiocarbohydrazide-silver proteinate technique - PBN phosphate buffer with NaCl - S spherical protoplasts - WGA wheat germ agglutinin     

Abnormal Cell Envelope Ultrastructure of a Saccharomyces Mutant with Invertase Formation Resistant to Hexoses

The most obvious morphological characteristic of Saccharomyces mutant FH4C cells is the tendency to form clumps (production of invertase and alpha-glucosidase by this mutant is highly resistant to repression by hexoses). This peculiar feature arises from the abnormal cell envelope ultrastructure of the mutant. Clumps are formed as a result of the failure of the cell wall of the bud to separate from that of the mother cell. The cell wall also shows irregular thickening. There are many cells with a doughnut shape and with small budlike protrusions. Abnormal septation and wall invagination into vacuoles give rise to cells of differing sizes and irregular profiles. Many vesicles, tubules, and coiled membranous bodies originate from invaginations of the plasmalemma. These structures are frequently observed in the cell wall or the periplasmic space. The cells of mutant FH4C growing in 0.2 M glucose, unlike parent strain 303-67, contain many mitochondria. Large numbers of glycogen deposits are also found in many cells of FH4C.     

在分化条件下甜菊愈伤组织分生区细胞超微结构研究

对甜菊（Ｓｔｅｖｉａｒｅｂａｕｄｉａｎａ）愈伤组织中尚未发生器官分化的分生细胞团进行了超微结构研究．结果表明,在器官分化条件下,愈伤组织中形成的分生区域的细胞体积小,细胞核大,核仁明显,且具核仁泡,部分细胞核中含有核内含物．大量小液泡分布在细胞的边周或散布于整个细胞中．液泡中通常含有陷入的细胞质成分和膜状物．部分液泡的形成与内质网膨大有密切关系．同时也观察到由内质网形成的多圈膜和双层膜包围细胞质成分的同心环结构．高尔基体及其小泡丰富,有时聚集分布在细胞某一区域．核糖体密集,有的聚集成多聚核糖体．因此,愈伤组织中分生区的细胞与分生组织中的液泡化和分裂的细胞类似．分生区细胞的另一明显特征是出现质膜内陷．推测这些超微结构特征可能反映了甜菊愈伤组织器官分化前的某些形态变化。     

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.     

On the ultrastructure of differentiating secondary xylem in willow

Summary Studies of differentiating xylem inSalix fragilis L. show the immediate cambial derivatives to be ultrastructurally similar. The Golgi apparatus is important at all stages of wall synthesis, possibly producing (amongst other substances) hemicellulose material which is carried to the wall in vesicles or multivesicular bodies. The endoplasmic reticulum also contributes one or more components to the developing wall: at some stages during differentiation the endoplasmic reticulum produces electron opaque bodies which appear to be guided towards the wall by microtubules. Compact structures formed from concentric membranes (myelin-like bodies) have been found joined to rough endoplasmic reticulum, but their presence is not explained.Two types of plasmalemma elaboration occur: invagination of the plasmalemma itself to form vesicles which may contain cytoplasmic material; and vesicles between the plasmalemma and cell wall which are the result of single vesicles or multivesicular bodies traversing the plasmalemma. Both systems provide a means for transporting cytoplasmic material across the plasmalemma.Microtubules have been seen associated with all vesicles derived from the cytoplasm which appear to be moving towards the wall. The presence of microtubules may generally be explained in terms of orientation of vesicles, even if they also happen coincidentally to parallel the supposed orientation of microfibrils in the wall itself. It is possible to resolve connections between the microtubules and the plasmalemma.     

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.     

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.     

Pollen development of Rondeletia odorata (Rubiaceae)

Pollen wall ontogeny of RONDELETIA: odorata was studied with transmission electron microscopy (TEM) and scanning electron microscopy (SEM) from tetrad stage until maturity. The ontogenetic sequence of wall development in RONDELETIA: follows, to some extent, the basic scheme in the angiosperms, i.e., development starts centripetally with the pro-columellae in a plasmalemma surface coating (primexine) at the early tetrad stage when the microspores are still enveloped by callose, until intine formation in young pollen grains. The main ontogenetical features of Rondeletia odorata pollen are (1) the very thin irregular foot layer, (2) development of a continuous layer of radially oriented membranous granular material under the thick endexine, (3) initiation of intine before first mitosis with characteristic radial plasmalemma invaginations, and (4) a strong stretching force upon engorgement just prior to dehiscence, which leads to reduction in thickness of all wall layers. The possible function of Golgi vesicles in the considerable increase in surface area of the plasmalemma at intine initiation is discussed. The endocingulum observed on acetolyzed and sectioned mature grains is explained ultrastructurally.