FINE STRUCTURE OF MEMBRANOUS AND MICROFIBRILLAR SYSTEMS IN THE CORTEX OF PARAMECIUM CAUDATUM

An electron microscope study of the cortex of Paramecium caudatum has revealed new details pertinent to several unresolved problems. The lateral boundaries of the alveoli do not regularly follow the crests of the polygonal ridges and thus their staining with silver cannot account for the external lattice seen by light microscopists. A granulo-fibrillar material is present, however, within the peaks of the ridges, which would account for the external lattice if so stained. Perforations are present between adjacent alveoli which make the whole mosaic of alveolar sacs within the cell's cortex continuous—both the membranes and the lumen. A microfibrillar system exhibiting a cross-striated pattern lies in the superficial cortex. These bands are inserted at their ends in the epiplasm and have a fine structure and arrangement suggesting a muscular function. The infraciliary lattice is a branching system of fibers with electron-opaque posts at the center of each branching locus. This system is distinct from the striated bands in morphology and in space. The epiplasm is discontinuous along the crests of the ridges, which may account for the pellicles' disposition to tear along these lines. A three-dimensional drawing is presented to show the interrelationships between the above membranous and microfibrillar systems.     

Secretory organelle docking at the cell membrane of Paramecium cells: Dedocking and synchronized redocking of trichocysts

We present the first evidence that secretory organelle docking at the cell membrane can be reversed in vivo. In nondischarge (nd) mutants of Paramecium tetraurelia all trichocysts can be detached from the cell surface within 2–3 h by different means, including cytochalasin B (but not D), high cell density, or Ca²⁺ ionophores. Considering the well-established ultrastructural differences between nd and wild-type (wt) cells, one can conclude that trichocyst docking at the cell periphery involves two docking sites (I, II): Site I ties the organelles to the epiplasm, and site II is the connection to the cell membrane at the fusogenic zone (expressed only in wt cells); both sites are close to the cell surface and only 150 nm apart. When the trigger for detachment of cortically docked trichocysts (high cell density, cytochalasin B) is relieved, trichocysts are synchronously reattached at the cell membrane, within 40–50 min, with a rate of 20–40 organelles/min, which far exceeds spontaneous docking rates. This is therefore also the first report on synchronization of secretory organelle docking. It is shown by radioactive leucine labeling that the same organelles are redocked, because trichocyst biogenesis is minimal under the conditions of de/redocking used. Surprisingly not only redocking but also detachment of trichocysts from the cell surface can be abolished by inhibitors of protein synthesis. Since Ca²⁺ ionophores mimic the effects of other conditions sufficient to detach trichocysts from the cell surface, we assume that a protein-dependent mechanism sensitive to Ca²⁺ (or other ions in exchange) may operate in trichocyst detachment. The precise mechanism involved in attachment or detachment of trichocysts remains to be elucidated.     

Formation of the plasma membrane during regeneration ofParamecium caudatum

Summary Fragments ofParamecium caudatum cells obtained by merotomy were fixed in 1% OsO₄ within 5 seconds after cutting. The ultrastructure of the damaged area of the fragment was studied in oriented ultrathin sections and by scanning electron microscopy. The cytoplasm exposed by merotomy was covered during a few seconds with a new membrane. This was a typical trilaminar membrane continuous with the plasma membrane covering the undamaged surface of the cell. The surface over the wound was wrinkled into irregular grooves and ridges. The cytoplasm, mitochondria and trichocysts in the injured region were electron translucent. The cytoplasm under the new membrane contained an unusually high amount of small membrane vesicles, 20–90 nm in diameter. These were probably the remnants of subpellicular alveoli and the plasma membrane destroyed by microsectioning. The possibility that the exposed cytoplasm would be covered by mere shifting of the existing plasma membrane can be excluded. The complex structure of the cortex with its subpellicular alveoli and regularly distributed cilia provide a strong argument against this notion. It seems probable that the new membrane was built up from the available molecular material,e.g., phospholipids and proteins present in the cytoplasm. Fragments of the membrane and alveolar membranes in the form of small vesicles may have also been included into the new membrane.     

Development of compound trichocysts in the ciliatePseudomicrothorax dubius

Summary During the logarithmic growth of the ciliatePseudomicrothorax dubius associations between mitochondria, rough endoplasmic reticulum and dictyosomes have been observed. The Golgi apparatus is very active and it is suggested that, as a consequence of cytotic activity, the contents of the Golgi vesicles become incorporated into large irregular vacuoles as globular material. The large vacuoles develop into trichocysts and the dictyosome derived globules consolidate to ultimately form the rod-like arms of the trichocysts of theMicrothoracidae.     

Electron microscopic studies of coated membranes in two types of gill epithelial cells of lamprey

Summary Coated membranes in two types of gill epithelial cell of adult lamprey, Lampetra japonica, were studied by electron microscopy. The type 3 gill epithelial cells possess well-developed microvilli or microfolds, apical vesicles and abundant mitochondria. The cytoplasmic surface of the microvillous plasma membrane is covered by a coat of regularly spaced particles with a center-to-center distance of about 15 nm. Each particle consists of a bulbous free end, about 10 nm in diameter, and a connecting piece, about 5 nm long. Apical vesicles are covered by a surface coat which consists of fine filamentous material but lack any special coating on their cytoplasmic surface.The type 4 cells (chloride cells) are characterized by apical vesicles, abundant mitochondria and cytoplasmic tubules. These tubules possess a coat on their luminal surface which consists of spirally wound parallel rows of electron-dense materials. The rows are about 16 nm apart and wound at a pitch of about 45°. The cytoplasmic surface of these tubules does not display a special coat. These coated membranes are assumed to be the sites of active ion transport across the plasma membrane. In particular, particles in type 3 cells and linear coat materials in chloride cells may be either loci of transport enzymes or energy generating systems. Apical vesicles lack any coating on their cytoplasmic surface but a fine filamentous coat is present on their luminal surface. They contain intraluminal vesicles and are continuous with apical ends of cytoplasmic tubules.     

Ultrastructure of the Fusion Area during Conjugation in the Suctorian Heliphrya erhardi (Rieder) Matthes*

SYNOPSIS. The suctorian Heliophrya erhardi (Rieder) Matthes is attached to the substrate by the flattened ventral side of the cell body. The dorsal is covered by a pellicle composed of 3 unit membranes. Below the pellicle is a 0.4–0.8-μm thick epiplasm composed of 6–8-nm thick fibrils. Microtubules form a network beneath the epiplasm. The epipalsm is penetrated by tube-like pellicular pits, which are lined by the cell membrane and end beneath the epiplasm in a saccule-like enlargement. During conjugation, 2 neighboring organisms form cytoplasmic processes which come into contact and fuse, thus forming a cytoplasmic bridge between the 2 cells. Around the bridge the pellicles of both organisms fuse, and the partners become united by a continuous common membrane system. Across the entire conjugation bridge the 2 fused epiplasms form a septum. Tube-like structures can be seen lying partly in the epiplasmic septum and partly in the adjacent cytoplasm. These structures are open at both ends and represent remnants of the pellicular pits. No trace of the original pellicular membranes can be found at the fusion area within the epiplasmic septum. The cytoplasm of the conjugation partners is separated only by the fused epiplasms forming the epiplasmic septum.     

Cytoskeleton,cell surface and the development of invasive plasmodial tapetum inTradescantia virginiana L.

Summary The anther tapetum inTradescantia virginiana L. is of the invasive plasmodial type: the cells lose their walls during early spore meiosis and develop long invasion processes which invade the loculus to penetrate spaces between the sporogenous cells. Fusion to form a syncytium is delayed and conventional ultra-thin sections and the Thiéry reaction reveal the presence of a loose fibrillar extracellular cell coat on the free surfaces of tapetal cells and their invasion processes. Cell fusion involves formation of apposition areas characterized by an absence of cell coat and the local appearance of microtubular arrays. Conspicuous membrane sacs, associated closely with microtubules, were found to migrate to and accumulate at the plasma membranes near the fusion sites and sporogenous cells. Microtubules are always present in the cortical regions of the tapetal cells and their invasion processes. It is surmised that microtubules are not responsible either for initiating or guiding tapetal invasion of the loculus; instead they may help to sustain the form of the invasion processes, help in the migration of membrane sacs, and participate in cell fusion. The cell coat disappears with syncytium formation towards the end of meiosis, and the developing spore cells become surrounded by a perispore membrane, which, derived from the original plasma membranes and augmented by membrane sacs, forms labyrinthine membrane reservoirs that are described further in the accompanying paper.     

Structure and development of the surface coat of erythrocytic merozoites of Plasmodium knowlesi

Summary The surface of extracellular merozoites of P. knowlesi is covered with a coat 15–20 nm thick, made up of clusters of filaments standing erect on the plasma membrane. Filaments have stems 2 nm thick, the peripheral ends of which are complex, branching or ending in long trailing threads. Coat filaments occur on the surface of the parasite in regular rows at an early schizont stage, and persist until well after merozoite release. They are sensitive to trypsin and papain, and bind ethanolic phosphotungstate, indicating a proteinaceous nature. They are also removed by exposure to phosphate-buffered saline. Filaments bear negative charges, binding cationised ferritin throughout the depth of the coat and staining with ruthenium red. They cover the whole merozoite surface and mediate intercellular adhesion at distances of 15–150 nm, membrane to membrane. It is suggested that these filaments correspond to a major merozoite surface protein, and are important in the initial capture of red cells.     

Spine-scale reorientation inApedinella radians (Pedinellales,Chrysophyceae): the microarchitecture and immunocytochemistry of the associated cytoskeleton

Summary Motile unicells ofApedinella radians have the extraordinary ability to instantaneously reorient six elongate spine-scales located on the cell surface. Extracellular striated fibrous connectors (termed microligaments) attach spine-scales to discrete regions of the plasma membrane underlain by intricate cytoplasmic plaques. A complex cytoskeleton is associated with the plaques and appears responsible for spine-scale movement. Three cytoskeletal proteins have thus far been identified by immunofluorescence using anti-tubulin, anti-actin, and anti-centrin. The three-dimensional configuration of the cytoskeleton has been established and consists of filamentous bundles of actin and centrin which form stellate systems interconnecting the plaques. Additionally, there is a network of microtubular triads which originate on the surface of the nuclear envelope and subtend the plasma membrane and also support several tentacular protrusions. It is proposed that contraction of the actin and/or centrin filamentous bundles is responsible for the reorientation of the spine-scales.     

Fine structure of the dorsal bristle complex and pellicle of Euplotes

The fine structure of the dorsal bristle complex and pellicle of non-developing Euplotes eurystomus is described in detail by scanning and transmission electron microscopy. The bristle-pit unit is a highly differentiated complex of organelles. The bristle complex is composed of a pair of kinetosomes (basal bodies) joined by a connective. The anterior kinetosome bears the bristle cilium, which contains a polarized network of particles (“lasiosomes”). The posterior kinetosome bears a very short, knob-like “condylocilium,” and has an associated striated fiber. Accessory ribbons of microtubules are also associated with the kinetosome couplets. Parasomal sacs, a septum connecting the bristle cilium to the anterior wall of the pit, core granules of the kinetosomes, and large membranous ampules are described. The organization of the bristle complex bears many similarities to the somatic ciliature of other ciliates. The pellicle of Euplotes is composed of a continucus outer cell membrane subtended by membranous alveoli, which contain a “fibrous mat.” Two sheets of subpellicular microtubules (longitudinal and transverse) are located just beneath the alveoli. The “epiplasm” seen in some other ciliates is apparently absent in Euplotes. The texture of the cell surface is a pattern of folds or rugae composed of the outer cell membrane and the upper membrane of the alveolus. The pattern of rugae probably defines the “silverline-system” of light microscopy.     

Secretory organelle docking at the cell membrane of Paramecium cells: dedocking and synchronized redocking of trichocysts

We present the first evidence that secretory organelle docking at the cell membrane can be reversed in vivo. In nondischarge (nd) mutants of Paramecium tetraurelia all trichocysts can be detached from the cell surface within 2-3 h by different means, including cytochalasin B (but not D), high cell density, or Ca2+ ionophores. Considering the well-established ultrastructural differences between nd and wild-type (wt) cells, one can conclude that trichocyst docking at the cell periphery involves two docking sites (I, II): Site I ties the organelles to the epiplasm, and site II is the connection to the cell membrane at the fusogenic zone (expressed only in wt cells); both sites are close to the cell surface and only 150 nm apart. When the trigger for detachment of cortically docked trichocysts (high cell density, cytochalasin B) is relieved, trichocysts are synchronously reattached at the cell membrane, within 40-50 min, with a rate of 20-40 organelles/min, which far exceeds spontaneous docking rates. This is therefore also the first report on synchronization of secretory organelle docking. It is shown by radioactive leucine labeling that the same organelles are redocked, because trichocyst biogenesis is minimal under the conditions of de/redocking used. Surprisingly not only redocking but also detachment of trichocysts from the cell surface can be abolished by inhibitors of protein synthesis. Since Ca2+ ionophores mimic the effects of other conditions sufficient to detach trichocysts from the cell surface, we assume that a protein-dependent mechanism sensitive to Ca2+ (or other ions in exchange) may operate in trichocyst detachment. The precise mechanism involved in attachment or detachment of trichocysts remains to be elucidated.     

Caveolin, a protein component of caveolae membrane coats.

Caveolae have been implicated in the transcytosis of macromolecules across endothelial cells and in the receptor-mediated uptake of 5-methyltetrahydrofolate. Structural studies indicate that caveolae are decorated on their cytoplasmic surface by a unique array of filaments or strands that form striated coatings. To understand how these nonclathrin-coated pits function, we performed structural analysis of the striated coat and searched for the molecular component(s) of the coat material. The coat cannot be removed by washing with high salt; however, exposure of membranes to cholesterol-binding drugs caused invaginated caveolae to flatten and the striated coat to disassemble. Antibodies directed against a 22 kd substrate for v-src tyrosine kinase in virus-transformed chick embryo fibroblasts decorated the filaments, suggesting that this molecule is a component of the coat. We have named the molecule caveolin. Caveolae represent a third type of coated membrane specialization that is involved in molecular transport.     

Cortical microtubules mark the mucilage secretion domain of the plasma membrane in Arabidopsis seed coat cells

During their differentiation Arabidopsis thaliana seed coat cells undergo a brief but intense period of secretory activity that leads to dramatic morphological changes. Pectic mucilage is secreted to one domain of the plasma membrane and accumulates under the primary cell wall in a ring-shaped moat around an anticlinal cytoplasmic column. Using cryofixation/transmission electron microscopy and immunofluorescence, the cytoskeletal architecture of seed coat cells was explored, with emphasis on its organization, function and the large amount of pectin secretion at 7 days post-anthesis. The specific domain of the plasma membrane where mucilage secretion is targeted was lined by abundant cortical microtubules while the rest of the cortical cytoplasm contained few microtubules. Actin microfilaments, in contrast, were evenly distributed around the cell. Disruption of the microtubules in the temperature-sensitive mor1-1 mutant affected the eventual release of mucilage from mature seeds but did not appear to alter the targeted secretion of vesicles to the mucilage pocket, the shape of seed coat cells or their secondary cell wall deposition. The concentration of cortical microtubules at the site of high vesicle secretion in the seed coat may utilize the same mechanisms required for the formation of preprophase bands or the bands of microtubules associated with spiral secondary cell wall thickening during protoxylem development.     

Biochemical Approaches to Problems of Cellular Patterning*†

SYNOPSIS. The expression of intracellular patterning is perhaps nowhere more impressive than in the arrangements of structural elements associated with the cell surface in protozoa. The view is proposed that biochemical studies of protozoan plasma membranes and associated surface structures represent important contributions of potential significance for the understanding of the perpetuation, and expression of positional information at the intracellular level. Some recent work dealing with the isolation, identification, and metabolism of pellicular proteins in Tetrahymena is presented and discussed. Some integral membrane proteins have been identified by iodination and polyacrylamide gel electrophoresis. Labeling studies suggest heterogeneous turnover rates within the group of presently identified membrane proteins. High molecular weight proteins with some similarity to spectrin have been isolated from Tetrahymena epiplasm. It is suggested that the ciliate epiplasm is one example of membrane-associated, actomyosin-like systems found in a variety of cell types. The epiplasm may play a role in the positioning of surface-associated structures and in the control of cell shape.     

Ultrastructural Features of Allantosoma intestinalis,a Suctorian Ciliate Isolated from the Large Intestine of the Horse1

ABSTRACT. Allantosoma intestinalis, a suctorian ciliate isolated from the intestine of the horse, was studied utilizing light and electron optical methods. These small sausage-shaped organisms have a varying number of tentacles (between one and 12) located at each extremity of the body. The microtubular axoneme of each tentacle in cross-section consists of two files of microtubules arranged in a daisy-like configuration. Haptocysts occur in the tentacle shaft, abutted to the plasma membrane of the knob of the tentacle, and in the cell body. The haptocysts are bottle-shaped, with prominent annular striations around their midportion. The cell is covered by three membranes, an outer plasma membrane, an outer alveolar, and an inner alveolar membrane. A thin epiplasmic layer is found beneath the inner alveolar membrane, and a single row of microtubules underlies the epiplasm. The subpellicular microtubules are arranged parallel to each other forming a corset around the cell along the long axis: such a system is not characteristic of suctorians. A field of diminutive kinetosomes (each 180 nm long, max. of 15 per field), lacking cilia, was found below the cortex. The function of these prokinetosomes is unknown. A ciliated swarmer has not been observed, only the nonciliated adult. The characteristics of Allantosoma are compared with those of other suctorian genera.     

Immunolocalization of the exocytosis-sensitive phosphoprotein, PP63/parafusin, in Paramecium cells using antibodies against recombinant protein

 We have localized a structure-bound fraction of the exocytosis-sensitive phosphoprotein, PP63/parafusin (PP63/pf), in Paramecium cells by widely different methods. We combined cell fractionation, western blots, as well as light and electron microscopy (pre- and postembedding immunolabeling), applying antibodies against the recombinant protein. PP63/pf is considerably enriched in certain cortical structures, notably the outlines of regular surface fields (kinetids), docking sites of secretory organelles (trichocysts) and the membranes of subplasmalemmal Ca²⁺-stores (alveolar sacs). From our localization studies we tentatively derive several potential functions for PP63/pf, including cell surface structuring, assembly of exocytosis sites, and/or Ca²⁺ homeostasis. Accepted: 4 December 1997     

Identification of caveolin-1 in lipoprotein particles secreted by exocrine cells

Caveolin-1 is a protein component (of relative molecular mass 22, 000) of the striated coat that decorates the cytoplasmic surface of caveolae membranes. Previous biochemical and molecular tests have indicated that caveolin-1 is an integral membrane protein that is co-translationally inserted into endoplasmic-reticulum membranes of fibroblast and epithelial cells such that its carboxy- and amino-terminal ends are in the cytoplasm. Here we identify caveolin-1 in the secretory pathway of exocrine cells. Secretion of caveolin-1 from pancreatic acinar cells and a transfected exocrine cell line, but not from Chinese hamster ovary cells, is stimulated by the secretagogues secretin, cholecystokinin and dexamethasone. The secreted caveolin-1 co-fractionates with apolipoproteins, indicating that it may be secreted in a complex with lipids.     

Colchicine inhibits plasmodium formation and disrupts pathways of sporopollenin secretion in the anther tapetum ofTradescantia virginiana L.

Summary During an earlier investigation, microtubules were observed at the periphery of invasion processes in the developing syncytial tapetum ofTradescantia virginiana L. They were also associated with membranous sacs that accumulate adjacent to tetrads, with putative fusion sites where the tapetal plasmodium is initiated, and, in postmeiotic stages, with the perispore membrane that encloses the developing spore cells. Colchicine was administered to developing flower buds to investigate the roles of these microtubules. The results indicate that microtubules neither initiate nor guide the tapetal invasion of the loculus. The treatments, however, resulted in absence of cell coat from invasion processes and prevention of cell fusion. They also inhibited polarized migration of membrane sacs and removed the associated microtubules. The development of an organized secretory apparatus at the perispore membrane was disrupted, with subsequent disordered deposition of sporopollenin in the extracellular spaces of the partially-fused plasmodium. The results suggest that microtubules participate in the formation and internal spatial organization of the tapetal plasmodium, and establishment of a secretory surface that normally produces sporopollenin at the tapetum-microspore interface.     

Studies on the surface coat of Paramecium aurelia

Summary The plasma membrane of Paramecium aurelia is covered with a ruthenium red stainable surface coat. Results obtained after digestion with pronase, trypsin and neuraminidase suggest the glycoprotein nature of this structure. Lipid extraction also affects the surface coat forming material. The results are consistent with the model proposed by Ginsburg and Kobata dealing with spatial configuration of the surface coat components.Authors are grateful to Mrs. D. Kucharczyk for very efficient technical assistence, to Mrs. Z. Kaminska for sectioning the material and Mr. A. Renski for help with the electron microscope service.     

The respiratory system of the genusCallithrix,the South American monkey

It has been described the cytology of the following parts of the respiratory system of some South American primates:Callithrix jacchus andCallithrix argentata melanura. The nasal cavities are divided into three parts: a vestibule, covered with a stratified nonkeratinized squamous epithelium; the respiratory portion, consisting of a pseudostratified columnar ciliated epithelium with goblet cells and the olfactory portion which is also covered with a high respiratory epithelium without goblet cells. The trachea is lined with a mucous membrane, whose epithelium is pseudostratified columnar ciliated with scarce goblet cells in the proximal portion unlike to the distal one. In the dorsal portion of the trachea, at the level of the gap between the two ends of incomplete cartilaginous rings, the epithelial lining is of transitional type. The incomplete hyaline cartilaginous rings present centers of calcification. The right and left lungs consist of two and three lobes respectively characteristic for these species, but they are not divided into lobules by connective tissue as in other ones. The bronchi, bronchioles and the respiratory portion, respiratory bronchioles, alveolar ducts, alveolar sacs, and alveoli present the typical respiratory structure with exception of their cartilaginous configuration; the cartilage continues as far as the respiratory bronchioles and alveolar ducts. These last structures are formed by a thin squamous epithelium, in which we observed two types of alveolar lining cells. This work was supported by grants from the Consejo Nacional de Investigaciones Cientificas y Técnicas (CONICET) and EHIGE program. Postgraduated fellow from CONICET. established Investigator and Director of EHIGE (Estudio Histológico comparado del Sistema de Glándulas Endócrinas) from CONICET.