Rhombic particle arrays in gill epithelium of a mollusc, Aplysia californica.

Gill epithelia from adult and juvenile Aplysia were examined by conventional thin section and freeze-fracture methods. Freeze-fracture replicas of adult gill epithelium revealed septate and gap junctions, which served as membrane markers for the epithelial cells. In these same cell membranes, non-junctional rhombic arrays of intramembranous particles were observed on prominent ridges on the membrane P fracture face of some epithelial cells. In thin sections of adult epithelium, nerve terminals were observed abutting the lateral plasma membranes near the basal lamina of some epithelial cells. Correlative areas of plasma membrane in freeze-fracture replicas showed a close association between rhombic particle arrays and abutting nerve terminals. In thin sections of juvenile Aplysia, nerve terminals abutting the epithelial cells were not recognizable, and rhombic arrays were not observed in freeze-fracture replicas. This suggested that a developmental association existed between the appearance of rhombic arrays in adult epithelia and their innervation. It is not known with certainty if, in invertebrates, rhombic arrays are an essential structural entity of all innervated cell membranes; however, in the cells thus far studied, there appears to be an associative condition. In the case of the gill epithelium of Aplysia, rhombic arrays are located in the same vicinity as the abutting nerve terminals. Similar arrays of intramembranous particles have been observed in myoneural postjunctional complexes of other invertebrates and have been interpreted to be the morphological expression of neurotransmitter receptors. An analogous explanation is put forth, namely that rhombic arrays may represent the structural correlates of neurotransmitter receptors and/or ionic channels in innervated membranes of invertebrates.     

Veils, mounds, and vesicle aggregates in neurons elongating in vitro.

“Veils” are thin membraneous expanses spread between growth cone microspikes of living and fixed cells. Some veils lack vesicular contents, while others contain membranous sacs, tubules, and vesicles. Mounds are bulges from the cell surface that are filled with vesicles; they are present on somas, along axonal surfaces, and on growth cones of fixed cells. Transmission electron microscopy (TEM) of whole, unsectioned cultured cells shows that, in many cases, the “vesicles” seen in thin sections are, in fact, sacs or tubular structures, twisting in complex ways within the interior of a mound. Veils and mounds have certain distinctive characteristics when compared with adjacent neuronal regions: Cortical microfilamentous material is lacking beneath the plasma membrane of both mounds and veils, as well as between the vesicular or tubular contents of these structures; and, as reported elsewhere [37], cationic ferritin does not bind to the outer surface of mounds and veils. Experiments done to determine if fixation has effects on mound formation suggest that the final morphology of mounds may be induced by glutaraldehyde fixation.     

External manifestation of plate-like particle arrays inthe plasma membrane of Tetrahymena

Plate-like arrays of 3–4 nm particles were previously reported to occur on the P face of the plasma membrane of Tetrahymena. Surface replicas have now been prepared from frozen, deep-etched cells. These replicas demonstrate that plate-like arrays are manifested on the external surface of the plasma membrane. This observation lends support to the idea that the plate-like arrays have a receptor function related either to feeding or to mating behavior in Tetrahymena.     

Electron Microscopy of Equine Infectious Anemia Virus

Equine infectious anemia (EIA) virus was observed in thin sections of infected cultured horse leukocytes by electron microscopy. The virus particles had a spherical shape and were between 80 and 120 nm in diameter. Most of them contained an electron-dense nucleoid 40 to 60 nm in diameter. They were observed to form by a process of budding from the plasma membrane and appeared to have thin surface projections. The particles described were not detected in uninfected cultured cells, and their appearance could be prevented by adding EIA immune serum to the inoculum. The implications of these findings in the classification of EIA virus are discussed.     

Particle Assemblies in the Plasma Membrane of Tetrahymena: Relationship to Cell Surface Topography and Cellular Morphogenesis1

During a freeze-fracture electron microscopical study of the plasma membrane of Tetrahymena, several different types of organized particle assemblies were observed. Three of these were found only on the protoplasmic face and were localized in the anterior-ventral region of the cell. These consisted of plate-like arrays composed of 4–25 triplet rows of small 3–4 nm particles; long, paired linear arrays localized at the tops of cortical ridges and composed of 7–8 nm particles; and elongated tetragonal arrays located in the grooves between ridges and composed of approximately 10 nm particles. The distribution of these arrays is consistent with roles in cellular morphogenesis, chemoreception, or cell-cell pairing during conjugation. In addition, a unique particle track associated with the cytoproct (anal pore) was observed in the external face of the plasma membrane. Furthermore, the protoplasmic face of the plasma membrane is characterized by a high density of particles organized into localized microarrays, consisting of small paracrystals or strings, which exhibit a loose higher-order patterning most evident toward the anterior end of the cell. Particle distributions on the protoplasmic face do not appear to be significantly altered by conditions that cause clumping of alveolar membrane particles. Taken together, these observations are consistent with the idea that the proteins of the plasma membrane are highly ordered and relatively immobile and that the structure of the plasma membrane is regionally differentiated.     

Cell walls,plasma membranes,and dictyosomes during zygote maturation ofClosterium ehrenbergii

Summary The cell wall formation and its correlation with the plasma membrane and dictyosome were investigated by an electron microscope in the zygote cells ofClosterium ehrenbergii. During zygote maturation, six wall layers were formed outside the plasma membrane. Wall layer III was the thickest layer and consisted of microfibril bundles. Dictyosomes produced flat vesicles during formation of wall layer III. Hexagonal arrays of rosette particles appeared in the plasma membrane in this period, thus confirming the simultaneous occurrence of flat vesicles and hexagonal particle arrays in the formation of microfibril bundles even at different stages of the life cycle. Wall layer VI was second in thickness and consisted of single microfibrils. Neither flat vesicles nor hexagonal particle arrays were observed during formation of this layer.     

Salt effects on membranes of the hypodermis and mesophyll cells of Avicennia germinans (Avicenniaceae): a freeze-fracture study

Freeze-fracture electron microscopy was used to investigate intramembranous particle (IMP) densities and particle distributions in the plasma membrane and tonoplast of the cells of secreting and nonsecreting leaves of Avicennia germinans (L.) Steam. Intramembranous particle densities of the protoplasmic (P) and exoplasmic (E) face of the plasma membrane and tonoplast were significantly higher in hypodermal cells of secreting leaves than of nonsecreting leaves. In contrast, no significant differences in the frequency of intramembranous particles were found in any membrane faces of secreting or nonsecreting mesophyll cells. However, particle densities were higher in the plasma membrane and tonoplast of the mesophyll cells, compared to the hypodermal cells, with the exception of the P-face of hypodermal plasma membranes of secreting tissue, which had the highest particle density measured. Particle distributions were dispersed and no discernible patterns such as paracrystalline arrays or other multi-IMP structures were observed. Results support the hypothesis that secretion is coupled to changes in membrane ultrastructure, and the possibility that salt secretion is an active process driven by integral membrane proteins such as the H⁺/ATPase. Additionally, the hypodermal cells of the leaf may function as storage reservoirs for salt as well as water, suggesting a regulatory role in salt secretion.     

Fine structures of sponge cell membranes: comparative study with freeze-fracture and conventional thin section methods

Freeze-fracture replicas of sponge cell membranes revealed in general a low density of intramembranous particles, with the exceptions of the membrane (silicalemma) surrounding the siliceous spicules in Ephydatia and the membranes of spherulous cells in Chondrosia. In addition, several types of particle arrangements were observed. A classical necklace is present at the base of the choanocyte flagellum. Rosettes of particles are particularly obvious in the apical membranes of choanocytes, where they are associated with the fuzzy coat covering these cells. Parallel ridges of particles were observed along the microvilli of the choanocyte collar, at sites of insertion of connecting filaments. Rows of particles were observed in the plasma membrane of pinacocytes in Ephydatia where they are located on areas deformed by protruding fibrillar inclusions. Pinacocyte plasma membranes in this species also can contain accumulations of particles which are likely related to desmosomes. Single rows of aligned particles and double rows of staggered particles (sometimes organized in large plates) in addition to rhombic particle arrays were encountered on replicas of marine sponge cell membranes. No classical arrangements corresponding to gap junctions, tight junctions or septate desmosomes were observed. The significance of these data is analysed.     

Differential extractability of influenza virus hemagglutinin during intracellular transport in polarized epithelial cells and nonpolar fibroblasts

Biochemical changes in the influenza virus hemagglutinin during intracellular transport to the apical plasma membrane of epithelial cells were investigated in Madin-Darby canine kidney (MDCK) cells and in LLC-PK1 cells stably transfected with a hemagglutinin gene. After pulse-labeling a substantial fraction of hemagglutinin was observed to become insoluble in isotonic solutions of Triton X-100. Insolubility of hemagglutinin was detected late in the transport pathway after addition of complex sugars in the Golgi complex but before insertion of the protein in the plasma membrane. Insolubility was not dependent on oligosaccharide modification since deoxymannojirimycin (dMM), which inhibits mannose trimming, failed to prevent its onset. Insolubility was not due to assembly of virus particles at the plasma membrane because insoluble hemagglutinin was also observed in transfected cells. Hemagglutinin insolubility was also seen in MDCK cells cultured in suspension and in chick embryo fibroblasts, indicating that insolubility and plasma membrane polarity are not simply correlated. In addition to insolubility, an apparent transport-dependent reduction of the disulfide bond linking HA1 and HA2 in hemagglutinin was detected. Because of the timing of both insolubility and the loss of the disulfide bond, these modifications may be important in the delivery of the hemagglutinin to the cell surface.     

Ultrastructural localization of concanavalin A receptors in the plasma membrane: association with underlying actin filaments

Whole-mount cell preparations of cultured rat 3Y1 cells were examined by stereo electron microscopy to identify the ultrastructural localization of concanavalin A (Con A) receptors in the plasma membrane, and to clarify the relationship between Con A receptors and cytoskeletal components. Well spread monolayer cells were extracted with saponin, briefly fixed, and then partially broken open with shearing force to facilitate the introduction of antibodies for identification of actin filaments. Stereo electron microscopy of such treated cells revealed a 3-dimensional image of filamentous structures such as fine filaments, microtubules (MT) and endoplasmic reticulum (ER) in the flattened areas of each cell. Just beneath the plasma membrane were meshworks of actin-containing fine filaments, as identified by an immunogold staining method. Microtubules and ER were observed to be either directly or indirectly associated with this meshwork. The broken open part of each cell exhibited a meshwork of filaments which were associated with the cytoplasmic surface of the plasma membrane. Some of the filaments were connected to the plasma membrane either by their ends or by their lateral surfaces. The localization of Con A receptors was examined by binding colloidal gold-labelled Con A to the surface of fixed, saponin-extracted cells. Virtually all gold particles bound externally at the same membrane sites where intracellular actin filaments attached internally. The observations strongly suggest that the distribution of Con A receptors was regulated by the underlying meshwork of actin filaments.     

A picornavirus-like pathogen of Cotylogaster occidentalis (Trematoda: Aspidogastrea), an intestinal parasite of freshwater mollusks

Nonoccluded, icosahedral picornavirus-like (PVL) particles, 23 nm in diameter, forming paracrystalline arrays were seen in the cytoplasm of various cells in Cotylogaster occidentalis. Viral inclusions were visible in live specimens and in sections prepared for light and electron microscopy. All worms examined over a 2-year period were found to be infected. Infections were naturally acquired and susceptibility was not associated with any particular developmental stages. Development of viral inclusions involved an increase in the inclusion volume, progressive accumulation and condensation of materials into the interior of the inclusions, and formation of multilamellar membrane networks. Virus particles were observed in the stroma of the inclusions in association with multilamellar spherical bodies. Mature PVL particles aggregated into polygonally shaped paracrystalline arrays. When such arrays occurred in the surface tegument, local disruption of the tegumentary membrane may liberate these particles into the environment. PVL particle production did not exhaust glycogen content of infected cells and did not appear to affect short-term survival of the parasite outside the molluscan host.     

Morphological observations on the formation and stability of the crystalline arrays in the plasma membrane of Saccharomyces cerevisiae

Two-dimensional crystalline arrays of freeze-fracture particles are known to occur in abundant quantities in the plasma membrane of stationary state yeast cells. Although these crystalline arrays are seen only infrequently in cells during mid-exponential growth, we now observe that formation of crystalline arrays can be induced in such cells by a “metabolic starvation” protocol. Surprisingly, starvation-induced formation of crystalline patches can be prevented by inhibition of new protein synthesis during the starvation period. The size and quantity of crystalline arrays can be increased by removal of the cell wall prior to starvation. Induction of crystalline arrays in protoplasts has made it possible to investigate the surface morphology of the crystalline particles in isolated membranes as well as at the extracellular surface of intact protoplasts. The stability of isolated crystalline arrays to several detergents has been investigated and conditions have been found that result in improved morphological purity of the isolated crystalline patches.     

STUDIES OF THE ULTRASTRUCTURE AND RIBOSOMAL ARRANGEMENTS OF THE PLEUROPNEUMONIA-LIKE ORGANISM A5969

Thin-section electron microscopy, together with isolation of cellular organelles by differential centrifugation and chemical analysis, has been used to investigate the ultrastructure of the avian pleuropneumonia-like organism A5969. Each cell (approximate diameter 5500 A) was surrounded by a 150 A plasma membrane. In the center of the cell was an unbounded area, granular in appearance and containing the cell's DNA. The periphery of the cell contained granules of several different sizes and densities. The most dense particles (150 A) corresponded to the 78S ribosomes. These particles exhibited two predominant arrangements: (a) sometimes they showed cubic packing; (b) most arrays, however, were consistent with cylindrical arrangements of approximately 50 particles. Bundles of up to 18 arrays were observed. Structured blebs have been found protruding from the surface of log phase cells.     

Orthogonal arrays of particles in the gill epithelium of the Atlantic hagfish,Myxine glutinosa

Summary Freeze-fracture replicas of hagfish gill epithelium revealed orthogonal arrays of 6-nm particles in the basolateral plasma membrane of pavement cells. The arrays consisted of 4–16 particles and occupied an area of 340±170 nm². These particle arrays, which are considered to be sites of ionic leakage, possibly contribute to the regulation of the pericellular micromilieu of adjacent mitochondria-rich cells.     

Stereo views and immunogold labeling of the pellicular microtubules at the inner surface of the plasma membrane of Leishmania as revealed by fracture-flip.

We used a modification of fracture-flip to reveal the nanoanatomy of the inner surface of the plasma membrane in promastigotes of Leishmania. After freeze-fracture, lightly fixed promastigotes were coated with a stabilizing layer of carbon evaporated from an electron gun, thawed, and washed. Fractured promastigotes attached to the carbon casts by the protoplasmic (i.e., inner) halves of their plasma membranes were treated with Triton X-100, followed by exposure to low concentrations of trypsin and thorough washing. This was followed by picking up and flipping of the replicas, followed by air-drying. The actual inner surfaces of the plasma membrane were then imaged by platinum shadowing. Extended, three-dimensional, high-resolution views of the inner surface of the plasma membrane showed parallel arrays of microtubules (average spacing 47 nm) closely apposed to the inner surface. Cytochemical labeling confirmed the morphological identification of both subpellicular and flagellar microtubules, as determined by treatment with mouse monoclonal anti-alpha- or anti-beta-tubulin, followed by labeling with goat anti-mouse IgG adsorbed to colloidal gold. Removal of the microtubules revealed parallel arrays of particles (average diameter 17 nm). We hypothesize that these particles represent the cytoplasmic portion of proteins that link the microtubules to the plasma membrane.     

Cell surface topography duringMarsilea spermiogenesis: Flagellar reorientation and membrane particle arrays

Summary Changes in the cell surface during spermiogenesis in the fern,Marsilea, have been investigated by freeze-fracture. Early in development 150 or more flagella appear on the surface of the spermatid cell. As they grow in length, they change orientation in relation to the spermatid cell surface and to each other. While the flagella are growing, a band of membrane particles surrounds each flagellum at the transition zone. These particles disappear near the end of development and are not seen in mature sperm. Other particles are associated with the plasma membrane during development. One set of particles is found early in spermiogenesis in hexagonal arrays. At the end of spermiogenesis, these are no longer observed, but clusters of particles, with no particular order, appear around the flagellar bases, following the line of the flagellar band.     

Surface Properties and Behavior of Lipid Extracts from Plasma Membranes of Cells Cultured as Monolayer and in Tissue-Like Conditions

The differences in the surface active properties of native lipids extracted from plasma membranes of cells cultured as a monolayer and in three-dimensional (3D) matrix were investigated. This experimental model was chosen because most of the current knowledge on cellular physiological processes is based on studies performed with conventional monolayer two-dimensional (2D) cell cultures, where cells are forced to adjust to unnaturally rigid surfaces that differ significantly from the natural matrix surrounding cells in living organisms. Differences between monolayer and 3D cells were observed in the lipid composition of plasma membranes and especially in the level of the two major microdomain-forming lipids—sphingomyelin (SM) and cholesterol, which were significantly elevated in 3D cells. The obtained results showed that culturing of cells in in vivo-like environment affected the surface active properties of plasma membrane lipids at interfaces which might influence certain membrane-associated interface processes. The detected differences in the lipid levels in 2D and 3D cell extracts affected significantly the behavior of the model lipid monolayers at the air–water interface (Langmuir monolayers) which resulted in different values of the monolayer equilibrium (γ_eq) and dynamic (γ_max, γ_min) surface tension and surface potential. Compensation of the SM content in extracts of 2D cell cultures up to a level close to the one measured in 3D cells approximated the monolayer properties to the values observed for 3D cells. These results implied that the interactions between the cells and the surrounding medium affected the level of plasma membrane SM and other lipids, which had a strong impact on the surface properties of lipid monolayers, such as γ_eq, γ_max, and γ_min, the compression/decompression curve shape, the hysteresis area during cycling of the monolayers, etc. We suggest that the elevated content of SM observed in plasma membranes of 3D fibroblasts could be responsible for an increased rigidity and possibly reduced permeability of cells cultured in 3D environment. The current results provide useful information that should be taken into account in the interpretation of the membrane physico-chemical properties of cells cultured under different conditions.     

Control of exocytotic processes: cytological and physiological studies of trichocyst mutants in Paramecium tetraurelia

The trichocysts of Paramecium tetraurelia constitute a favorable system for studying secretory process because of the numerous available mutations that block, at various stages, the development of these secretory vesicles, their migration towards and interaction with the cell surface, and their exocytosis. Previous studies of several mutants provided information (a) on the assembly and function of the intramembranous particles arrays in the plasma membrane at trichocyst attachment sites, (b) on the autonomous motility of trichocysts, required for attachment to the cortex, and (c) on a diffusible cytoplasmic factor whose interaction with both trichocyst and plasma membrane is required for exocytosis to take place. We describe here the properties of four more mutants deficient in exocytosis ability, nd6, nd7, tam38, and tam6, which were analyzed by freeze-fracture, microinjection of trichocysts, and assay for repair of the mutational defect through cell-cell interaction during conjugation with wild-type cells. As well as providing confirmation of previous conclusions, our observations show that the mutations nd6 and tam6 (which display striking abnormalities in their plasma membrane particle arrays and are reparable through cell-cell contact but not by microinjection of cytoplasm) affect two distinct properties of the plasma membrane, whereas the other two mutations affect different properties of the trichocysts. Altogether, the mutants so far analyzed now provide a rather comprehensive view of the steps and functions involved in secretory processes in Paramecium and demonstrate that two steps of these processes, trichocyst attachment to the plasma membrane and exocytosis, depend upon specific properties of both the secretory vesicle and the plasma membrane.     

STRUCTURAL FEATURES OF MESOSOMES (CHONDRIOIDS) OF BACILLUS SUBTILIS AFTER FREEZE-ETCHING

Freeze-etched cells of Bacillus subtilis have been studied with the electron microscope. The outer surface of the plasma membrane, i.e. the side facing the cell wall, is covered with numerous granules and short strands, each measuring approximately 50 A in diameter. These strands are occasionally seen to enter the cell wall. The inner surface of the plasma membrane, i.e. the side facing the cytoplasm, appears to be sparsely dotted with small particles measuring about 50 A. The envelope of mesosomes differs from the plasma membrane. Blunt protrusions arise from its outer surface; the inner surface appears smooth. Stalked particles, as described by other investigators after negative staining with phosphotungstic acid, were not observed on any membrane surface in our material. Preparations were also made of specimens prefixed in osmium tetroxide prior to freeze-etching. Under these conditions the bacterial membranes appeared to be surprisingly well preserved. In contrast to directly frozen, unfixed cells, some osmium tetroxide-fixed preparations showed a differentiation in cytoplasm and nucleoplasm, which made it possible to observe the close association of the mesosome with the latter.     

Similarity of junctions between plasma membranes and endoplasmic reticulum in muscle and neurons

The structure of membranes at junctions between the plasma membrane and underlying cisterns of endoplasmic reticulum in amphioxus muscle and mouse cerebellar neurons was studied using the freeze-fracture technique. In amphioxus muscle, subsurface cisterns of sarcoplasmic reticulum form junctions with the surface membrane at the level of the sarcomere I bands. On the protoplasmic leaflet of the sarcolemma overlying these junctions were aggregates of large particles. On the protoplasmic leaflet of the membranes of cerebellar basket, stellate and Purkinie cells there were similar aggregates of large particles. In both tissues, the corresponding external membrane halves had arrays of pits apparently complementary to the aggregates of large particles. Cross fractures through junctions showed that the particle aggregates in neuronal and muscle membranes were consistently located over intracellular cisterns closely applied to the plasma membrane. Thus, a similar plasma membrane specialization is found at subsurface cisterns in mammalian neurons and amphioxus muscle. This similarity supports the hypothesis that subsurface cisterns in neurons, like those in muscle, couple some intracellular activity to the electrical activity of the plasma membrane.