A freeze-etch study of the ultrastructure of red algal pit plugs

Summary Freeze-etch studies indicate that the fully-formed pit plug ofPalmaria palmata consists of a homogeneous plug core, a pair of plug caps, and associated membranes. A single cap membrane separates the two layers of each plug cap and is the only membrane found between the cytoplasm and the plug core. Due to the apparent junction of the plasmalemma and cap membranes, the plug core, along with the adjacent plug cap layer on either face of the core, is entirely membrane-bounded and essentially extracellular. Fracture faces of the cap membrane bear large populations of particles which are inconspicuous due to their low profile. The plasmalemma lining the plug core from cell to cell has numerous, very obvious particles on its fracture faces, more even than does the cytoplasmic plasmalemma.The relationship of the rhodophycean pit plug to the fungal septal pore plug is discussed.This report represents a portion of a thesis submitted in partial fulfillment of the requirements of a Master of Science degree, Cornell University, Ithaca, N.Y.     

Decoration of specific sites on freeze-fractured membranes

Fracturing under ultrahigh vacuum (UHV, P less than or equal to 10(-9) Torr) produces membrane fracture faces devoid of contamination. Such clean surfaces are a prerequisite for studies of interactions between condensing gases and distinct regions of a surface. For the study of water condensation, a device has been developed which enables production of pure water vapor and controlled variation of its partial pressure in an UHV freeze-fracture apparatus. Experiments with yeast plasmalemma fracture faces, produced at -196 degrees C and exposed to pure water vapor before replication, resulted in a "specific decoration" with ice crystals of those pits in the extraplasmic face where the corresponding particles of the plasmic face had been removed. Because water condenses as discrete ice crystals which resemble intramembrane particles, ice crystals might easily be misinterpreted as actual membrane structures. At low specimen temperature (T less than or equal to 110 degrees C) the structural features of membrane fracture faces produced under high vacuum (P approximately 10(-6) Torr) should, therefore, be interpreted with caution.     

Analysis of the structure of intramembrane particles of the mammalian urinary bladder

The luminal and discoid vacuole membranes of the superficial cell layer of the transitional epithelium of the mammalian urinary bladder have been studied by thin-sectioning and freeze-fracture-etch (FFE) electron microscope methods. For the FFE studies membranes were deposited on a cationized glass surface, covered by a thin copper disc, and fractured under liquid N2. Specimens were etched at -100 degrees C and replicated at -190 degrees C. A model of the lattice membrane derived from thin sections was used to predict the heights of the fracture faces above the glass surface. A hexagonal pattern of globular intramembrane particles spaced 160 A apart was seen in the external fracture (EF) face plaques as previously described and regarded as the dominant structure. However, very extensive areas of another pattern, seen before in only limited areas, have beeen found in the EF faces. The pattern consists of a smooth hexagonal lattice with the same space constant as the globular one but a different structure. By image analysis it consists of overlapping domains bordered by shared but incomplete metal rims. Each domain has a central spot of metal encircled by a shadow. The surface of the smooth lattice is partly complementary to the corresponding protoplasmic fracture (PF) face which shows a similar hexagonal lattice with the same space constant. The height of the smooth EF lattice above the glass substrate is the same as the plane of the center of the lipid bilayer predicted by the model. The mean heights of the particles of the globular EF lattice are greater than the total thickness of the membrane as predicted by the model and confirmed by measurements. The globular EF lattice is not complementary and it is concluded that the globular particles do not exist in the native membrane but arise artifactually during the preparatory procedures.     

Structural Changes in Membranes of Synchronized Cells demonstrated by Freeze-cleavage

FREEZE-CLEAVAGE is a new technique for studying the ultra-structure· of biological membranes, which fractures cell membranes in half, exposing two intramembranous fracture faces^1–3: the outer fracture face (OFF) and the inner fracture face (IFF). These fracture faces are partially covered with 70 Å globular particles which are thought to be unique structural components of cell membranes, formed by the association of membrane glycoproteins and lipids⁴. The 70 Å particles are dynamic structures and rapidly increase in density in the membranes of lymphocytes following exposure to mitogenic plant proteins (Scott and Marchesi, unpublished work).     

Freeze-fracturing in normal vacuum reveals ringlike yeast plasmalemma structures

The fine structure of the regular arrays of subunits seen on both plasmalemma fracture faces in resting and starved Saccharomyces cerevisiae (baker's yeast) has been compared using different freeze-fracture replication methods. Freeze-cleaving was carried out at 173 degrees, 133 degrees, and 108 degrees K under a vacuum of 2 X 10(-7) torr (2.6 X 10(- 7)mbar) or under liquid nitrogen at atmosphereic pressure. Independent of the preparation conditions (fracturing temperature, and whether cleaved under vacuum or liquid nitrogen), resting and starved yeast show a significant difference in the morphology of the subunits forming the regular arrays. The regularly arranged particles of the P face of the plasmalemma of starved yeast have a clear craterlike structure which has previously been reported to be demonstrated only by freeze-etching at very low temperatures in ultrahigh vacuum. A complementary structure is seen on the plasmalemma E face. Prolonged exposures of fracture faces under the protection of liquid nitrogen-cooled shrouds have shown that, because of the consequent drastic reduction of condensable gases in the specimen area, no detectable condensation contamination of exposed fracture faces occurs within 15 min at a specimen temperature of 108 degrees K. This shows that a complicated ultrahigh vacuum technology is not required for high resolution freeze- etching.     

Ultrastructure of the pellicle of Euglena gracilis

Deep-etching technique was used to investigate the organization of the pellicle complex of Euglena gracilis. The interpretation of the images was further supported by SEM and TEM investigations. Our results mainly validate data obtained by previous freeze-fracture studies on the E and P faces of the outer cortical membrane. At the level of the ridges, the outer E fracture face is highly organized in a regular striated pattern, whereas the P inner face shows a particulate structure. However, our images reveal that this particulate organization of the P face is not limited to the ridges, but it is displayed also by the grooves. Moreover, this face shows two distinct layers, a particulate layer facing the cytoplasm and a striated layer facing the E face; these layers represent different true fracture levels of the same P face.     

A new cell wall structure in a symbiotic and a free-living strain of the blue-green alga genus Chroococcidiopsis (Pleurocapsales)

Freeze etching studies in a symbiotic and a freeliving strain of Chroococcidiopsis revealed a specific layer in the outer cell wall not described so far from Cyanophyta. The layer showed a complex organisation: The main unit are ribbons, 2–3 nm thick, striated at right angle to the longitudinal axis. They are interwoven to a patchwork-like leaflet. The ribbons are virtually composed of globular particles associated in parallel rows. The cytoplasmic membrane and the cell walls of the symbiotic and the free-living strain were compared.Abbreviations cm cytoplasmic membrane - CW 1,2,3 cell wall layer 1,2,3 - EF exoplasmic fracture face - PF protoplasmic fracture face     

Fracture Faces in the Cell Envelope of Escherichia coli

Freeze-fracturing of Escherichia coli cells in the presence of 30% (v/v) glycerol resulted in a double cleavage of the cell envelope exposing two convex and two concave fracture faces ([Formula: see text], [Formula: see text] and [Formula: see text], [Formula: see text]) with characteristic patterns. Complementary replicas revealed the relationship of the fracture faces to their corresponding fracture planes. The inner fracture plane splits the plasma membrane at one particular level. Apparently the outer fracture plane was located in the outer part of the wall, as it was separated by a layer ([Formula: see text]) from the fractured profile (CW1) presumably corresponding to the murein layer. The outer fracture plane did alternate toward the cell periphery, exposing complementary smooth areas ([Formula: see text] and [Formula: see text]). When cells were freeze-fractured in the absence of glycerol, the outer cell surface appeared as an etching face rather than a fracture face. A schematic representation of the relative location of the different fracture faces in the E. coli cell envelope is given.     

Plasmodium falciparum: freeze-fracture of the gametocyte pellicular complex

Freeze-fracturing has been used to study the architecture of the pellicular complex of the gametocytes of Plasmodium falciparum. The gametocyte is surrounded by three membranes and a layer of subpellicular microtubules. During freeze-fracturing, each of the three membranes is split along its hydrophobic interior to yield a total of six fracture faces. The most obvious feature of each fracture face is the presence of globular intramembranous particles on their surfaces. The six fracture faces differ from one another in arrangement, size, and density of these intramembranous particles. In gametocytes, unlike in sporozoites, the intramembranous particles are always distributed randomly and lack any definite pattern or orientations. A unique feature of gametocytes revealed by the freeze-fracturing technique is the presence of several transverse sutures on the middle membrane that encircle the gametocyte and give it a segmented appearance.     

Plasmodium berghei: Architectural analysis by freeze-fracturing of the intraoocyst sporozoite's pellicular system

The technique of freeze-fracturing has been used to study the architecture of the pellicular complex of the intraoocyst sporozoite of Plasmodium berghei. The sporozoite is surrounded by three plasma membranes and a layer of subpellicular microtubules. During freeze-fracturing, each of the three membranes can split along its hydrophobic interior to yield a total of six fracture faces. The most obvious feature of each fracture face is the presence of globular intramembranous particles on the surface. The six fracture faces differ from one another in arrangement, size, and density of these intramembranous particles. Two of the fracture faces exhibit a unique arrangement of particles in well-organized parallel rows along the long axis of the sporozoite. This arrangement has not been reported in either the erythrocytic or the exoerythrocytic forms of Plasmodium spp. Another unique feature in the sporozoite revealed through freeze-fracturing is a single suture line that traverses the long axis of the inner two membranes of the parasite.     

The Fine Structure of Conidia of Botryodiplodia ricinicola with Observations on Effects of Chilling

Conidia of Botryodiplodia ricinicola (Saccardo) Petrak havebeen studied, principally by freeze-etch electron microscopy.Freshly harvested conidia have a thin scaly surface layer, freeof rodlets, which covers an otherwise homogeneous-looking wallwhich is continuous with the single centrally-perforate septum.The contours of the plasmalemma are usually smooth. Nuclei andsmall vacuoles are numerous. Hydrophobic fracture faces of theplasmalemma, tonoplasts and nuclear membranes variously revealintra-membrane particles or corresponding depressions or both.Lipid inclusions are small and numerous. Compact orderly stacksof membranes are present, sometimes one in each locule of theconidium. Conidia of a strain insensitive to chilling were seento differ only in respect of the distribution of intra-membraneparticles on fracture faces of tonoplasts. Chilled and chilled-and-soakedconidia of the wild type showed fine-structural differencesfrom untreated conidia, most obviously in respect of the greatersize of some of the lipid inclusions, but also in respect offeatures of the plasmalemma which after chilling contained plasmalemmasomesand, after subsequent immersion for 15 min, showed annular depressions.Also, intra-membrane particles in some membrane systems showedaltered distribution between the two hydrophobic fracture faces.It is concluded that cell lipids and cytoplasmic membrane systemsmay be involved in the previously demonstrated chilling sensitivityof conidia of this species. Botryodiplodia ricinicola, conidia, ultrastructure, chilling effects     

An in situ Freeze-Fracture Study of Spiroplasma citri and the Corn Stunt Spiroplasma

Spiroplasma citri and the corn stunt spiroplasma in sieve cells of Catharanthus roseus were investigated using freeze -fracture electron microscopy. Only the particle studded fracture faces of the plasmalemma could be exposed and not the surfaces of both the extraplasmatic and the plasmatic leaflet. The extraplasmatic fracture face (EF) shows a lower particle density than the plasmatic fracture face (PF). On the PF particle free areas could be observed, which are helically arranged in helical filaments. We suppose that the cytoplasmic fibrils, probably involved in motility processes and in maintaining the helical shape, underly the particle free area only.     

The Structure of the Canal Cell in the Style of Lilium regale

The fine structure of canal cell in the style of Lilium regale has been observed under light and electron microscopes by OMA thin section method and ultra-thin section method respectively. The ultrastructural specialization of the canal cells during their functional stages may be characterized as follows: 1. The cell wall on the secretory face of the canal cell has numerous branched ingrowths extending into the cytoplasm, and the plasmalemma closely follows the contours of the ingrowths to form the wall-membrane apparatus. This pattern of distribution of plasmalemma increases the surface-volume ratio of the cell to facilitate the secretion of solutes out of the cell. 2. The cell wall under the thin layer of cuticle on the outside of the secretory face is digested starting from the outer part and gradually extending to the inner part to form a large space, the temporary secretory layer. During the secretion of products by the cell, the thin layer of cuticle becomes ruptured in many places and finally disappeared. Therefore the cell wall of the secretory face remains a thin layer only at that time. The change of the layers of the cell wall is involved in the mechanism of cell secretion. 3. The ultrastructural characteristics of the canal cell indicate that this cell is active in synthesis, intercellular transport and energyn metabolism. Some of the major facts seen in all cases included the highly lobing of nucleus, abundance of endoplasmie reticulum throughout the cytoplasm and well developed mitochondria, dictyosomes and polysomes. During the secretory stage of the cell, mitochondria apparently concentrate near the wall-membrane apparatus. 4. There are numerous granular and vesicular structures near the wall-membrane apparatus on the secretory face, especially at the space between wall ingrowths and plasmalemma. The presence of these granular and vesicular structures is thought to be related to the secretory function of the cell. According to the specialized characteristics the canal cell is evidently a typical transfer cell of the secretory type.     

William Trager: An Appreciation

SYNOPSIS. Additional information on host interactions with trypanosomatid membranes was obtained from studies of a monomorphic strain of Trypanosoma brucei harvested at peak parasitemia from intact and lethally irradiated rats. Pellets of trypanosomes were fixed briefly in glutaraldehyde and processed for thin section electron microscopy or freeze-cleave replicas. Observations of sectioned material facilitated orientation and comparison of details seen in replicas. Fracture faces of cell body and flagellar membranes as well as 3-dimensional views of the nuclear membrane were studied. Cell body membranes of 80% of the organisms from intact rats contained random arrays of intramembranous particles (IMP). Aggregated clusters of particles appeared on the fracture faces of 20% of the trypanosomes. Some of these membranes had nonrandomly distributed particles aligned in distinct rows on the outer fracture face of both cell body and flagellum. Many inner face fractures of the cell body membranes had a particle arrangement similar to the longitudinal alignment of cytoskeletal microtubules. No aggregated particle distribution was seen in membranes of trypanosomes harvested from lethally irradiated rats. Replicas of trypanosome pellets also had plasmanemes as a series of attached, empty, coated membrane vesicles. These structures were found in close association with, as well as widely separated from the parasites. The shedding of these vesicles and the variation of particles in cell body membranes are discussed in light of antibody-induced architectural and antigenic changes in surface properties of trypanosomatids. The convex face of the inner membrane of the nucleus also is covered with randomly arrayed particles. More IMP were observed on the inner than on the outer nuclear membranes. Images of nuclear pores were also seen. The importance of these structures in drug and developmental studies of trypanosomes is discussed. On fracture faces of the flagellar membrane there were miniature maculae adherentes, unique to the inner fracture face and occurring only at regions of membrane apposition between cell body and flagellum. Each cluster of particles exposed by the freeze-cleave method corresponds to an electron-dense plaque seen in thin section images. However, because of a unique fracture pattern, these plaques were not revealed on the apposing body membranes, as illustrated in thin sectioned organisms.     

Ultrastructure of the Bacteroides nodosus cell envelope layers and surface.

The surface structure and cell envelope layers of various virulent Bacteroides nodosus strains were examined by light microscopy and by electron microscopy by using negative staining, thin-section, and freeze-fracture-etch techniques. Three surface structures were described: pili and a diffuse material, both of which emerged from one or both poles of the bacteria (depending on the stage of growth and division), and large rodlike structures (usually 30 to 40 nm in diameter) associated with a small proportion of the bacterial population. No capsule was detected. The cell envelope consisted of four layers: a plasma membrane, a peptidoglycan layer, an outer membrane, and an outermost additional layer. The additional layer was composed of subunits, generally hexagonally packed with center-to-center spacing of 6 to 7 nm. The outer membrane and plasma membrane freeze-fractured through their hydrophobic regions revealing four fracture faces with features similar to those of other gram-negative bacteria. However, some unusual features were seen on the fracture faces of the outer membrane: large raised ring structure (11 to 12 nm in diameter) on cw 3 at the poles of the bacteria; complementary pits or ring-shaped depressions on cw 2; and small raised ring structures (7 to 8 nm in diameter) all over cw 2.     

Restriction of glycolipids to the outer half of a plasma membrane: Concanavalin A labeling of membrane halves in acanthamoeba castellanii

Fracture-label, a method that permits the cytochemical characterization of faces produced by freezefracture, was used to determine the partition and distribution of a glycolipid on membrane fracture faces of Acanthamoeba castellanii cells. After treatment with concanavalin A (Con A), the glycolipid (a lipophosphonoglycan, LPG) was labeled with colloidal gold coated with horseradish peroxidase. The label was abundant over exoplasmic fracture faces (face E) of plasma membranes, but absent from protoplasmic fracture faces (face P). We conclude that, in A. castellanii, glycolipid molecules are restricted to the outer half of the plasma membrane. This conclusion is confirmed by experiments with cells disrupted by freezing and thawing, where access of label to the cell interior did not result in labeling of the inner surface. Our results establish the exclusive localization of a glycolipid to the outer half of a plasma membrane. Fracture-label is proposed as a new technique to investigate the distribution and partition of glycolipids in plasma and intracellular membrane halves.     

Isolation and properties of the plasmalemma in yeast

Summary A method is described for the isolation of fragments of the plasmalemma based on differential and density gradient centrifugation using cell free extracts from anaerobically grown Saccharomyces cerevisiae. Electron microscopically investigated frozen-etched specimens of isolated plasmalemma revealed the presence of globular particles attached to the outer surface of the membrane; these particles correspond to those observed in situ.In isolated plasmalemma a high specific activity of Mg⁺⁺-dependent ATPase, which is not sensitive to Oligomycin, is present. Yeast plasmalemma contains protein, lipids (including phospholipids) and an appreciable amount of polysaccharide. Hydrolysis of this polysacharide yields only mannose.The treatment of the isolated plasmalemma with detergents liberates the globular particles which can be isolated by density gradient centrifugation. Protein and polysaccharide occur in the respective fraction; therefore the globular particle represents a mannan-protein. It is concluded that the particles, which cover the plasma-membrane of plant cells, represent glycoproteins, that is, building stones to be incorporated into the fibrillar network of the cell walls.     

Organization of the cell membrane inEuglena

Summary The cell membrane ofEuglena gracilis has been investigated with the freeze-fracture technique. When split, this membrane produces two fracture faces which are striking in their non-complementarity. The P fracture face is covered with a high density of 110 Å (average diameter) particles, while the E face is made up of a complex series of striations occurring at regular angles to the pellicle ridges which encircle the organism. Under certain conditions, however, the structure of the P fracture face assumes a more ordered configuration, and striations are visible on this fracture face which are precisely complementary to those observed on the E face. These observations suggest that the cortical cell membrane ofEuglena may be organized along the lines of a two dimensional crystal. However, this pattern of organization is restricted to the cortical region of the cell membrane; as the membrane invaginates near the anterior end of the cell the fracture faces change abruptly, and organization more typical of other cell membranes is observed. This invagination forms an extensive reservoir in the anterior of the cell, and the membrane bounding it is distinctly fluid in structure, with clear examples of endo- and exocytosis observable. These differences suggest that the cell membrane inEuglena is divided into two distinct but contiguous regions, each specialized with regard to structure and function.     

Freeze-fracture studies on the cockroach hemocyte membrane complex: Symmetric and asymmetric membrane particle distributions

Summary Freeze-fracture studies were conducted on the membranes of normal cockroach hemocytes. The plasmalemma is asymmetric with the A fracture face containing 80–100 Å membrane intercalated particles at a concentration of 2500/². The B fracture face contains 120–150 Å particles with a relatively low density (800/²). The nuclear envelope displays an asymmetry with the A fracture face containing 1500 particles/² and the B face containing 300/ ². No significant particle size differences were observed in nuclear envelope fracture faces. Two types of symmetric membranes were also found in these cells. Both A and B fracture faces of the membrane surrounding the numerous cytoplasmic inclusion bodies contain particle sizes and concentrations similar to the B face of the plasmalemma. A second type of symmetry was observed in cells apparently engaged in exocytosis. Vesicles (0.1 D) from this process were completely particle free on both fracture faces. Such particle free vesicles could be found in the cytoplasm, attached to the plasmalemma, or completely separated from the cell.Supported by a Pharmaceutical Manufacturers Association Foundation Fellowship.The author wishes to thank Ms. Annalena K. Charla for assistance in plate preparation, Dr. Julius Schultz and the Papanicolaou Cancer Research Institute for use of the freeze-etch device, and Dr. David Smith for the electron microscope facilities.     

Ultrastructure and development of oil cells in Laurus nobilis L. leaves

The oil cell development in Laurus nobilis leaves has been studied. At the early developmental stage, when the cell wall consists of the outer cellulose wall only, the oil cells differ from the neighbouring mesophyll cells in their larger size, lower starch content and in their plastid organization. After the deposition of the lamellated suberin layer and the inner cellulose layer, a wall protuberance (cupule) is formed on the periclinal wall facing the epidermis. From its reaction with periodic acid-hexamine-silver nitrate, it is suggested that the cupule is cellulosic. The portion of the inner cellulose wall layer bearing the cupule seems to contain patches of suberin. Plasmodesmata occur in special wall protuberances and appear to become occluded with age. The oil produced inside the protoplast is secreted to the outside of the plasmalemma, and accumulates as a drop at the place predetermined by the cupule. Except at the cupule, the oil drop is surrounded by the plasmalemma.