Freeze-fracture Technique Research Articles

Microvilli-like Structures Secreting Chitin Crystallites

Vestimentiferans are good models for investigating the chitin secretion process. These worms, especially the giant tube worm Riftia pachyptila, inhabit a chitinous tube which results from the secretion of differentiated cells, associated in specific chitin secreting systems. Such systems were analyzed by means of transmission electron microscopy using both standard stained thin sections and freeze-fracture techniques. The cells exhibit original microvilli-like structures, that were named "cups," in relation to their shape. These specialized microvilli are thought to be involved in the chitin crystallite secretion. We describe the main features of these subcellular structures and the characteristics of the crystallites. A three-dimensional model of these organelles is suggested. We discuss the specificity of this crystallite synthesis and propose a tentative model of the microfibril formation. These findings lead us to hypothesize that the β chitin microfibril secretion is a highly regulated process, the cup-shaped structure being the first organelles found which may be involved in the chitin crystallite formation of multicellular organisms.

Calcium channel antagonist omega-conotoxin binds to intramembrane particles of isolated nerve terminals

Voltage-sensitive calcium channels play a key role in evoked neurotransmitter release and their distribution in presynaptic membranes can be critical for fast signalling at chemical synapses. Using a biotinylated derivative of the neuronal calcium channel antagonist, omega-conotoxin, and a combination of colloidal gold labeling and freeze-fracture techniques, we have labeled calcium channels present at the membrane of nerve terminals isolated from the electric organ of Torpedo marmorata. The biotinylated blocker exerts an inhibitory action on the high potassium-evoked release of adenosine triphosphate as the native toxin does and its interaction with biological membranes is specific as shown in displacement experiments. This study shows that an antagonist specific for voltage-activated calcium channels binds to intramembrane particles in presynaptic membranes, reinforcing the idea that these particles, concentrated at neurotransmitter release sites, effectively represent calcium channels.

Wuchereria bancrofti: Freeze-Fracture Study of the Epicuticle of Microfilariae

The freeze–fracture technique was used to analyze the surface of microfilariae of Wuchereria bancrofti. The examination of thin sections showed that the epicuticle is formed by two closely apposed membrane-like structures, appearing as heptalaminated structures. Freeze–fracture replicas revealed the presence of two fracture faces. The Outer one is smooth but presents rosette-like structures. The inner face, which is in contact with the hypodermal cells, presents a large number of particles.

Plasma membrane alterations of maturing goat (Capra indicus) spermatozoa: lectin-binding and freeze-fracture study.

A qualitative and quantitative analysis of lectin-binding sites has been undertaken on spermatozoa recovered from different regions of the epididymis of the goat (Capra indicus) using fluorescein isothiocyanate-linked lectins (Bauhinia purpurea BPA, Concanavalin A Con A, Dolichos biflorus DBA, Maclura pomifera MPA, Arachis hypogaea or peanut agglutinin PNA, Glycine max or soyabean agglutinin SBA, Ulex europaeus UEA, and Triticum vulgaris or wheat-germ agglutinin WGA), in conjunction with scanning and transmission electron microscopy, and freeze-fracture techniques. Flow cytometric analysis has also been used to quantitize binding affinity. Spermatozoa from caput to cauda epididymidis show no significant variation in lectin-binding ability, but the samples removed from the corpus epididymidis contain a greater number of binding sites. The passage of spermatozoa through the epididymidis is accompanied by a redistribution of the plasma membrane lectin-receptors covering the sperm head and tail. Receptors for BPA, DBA, PNA and SBA are specifically restricted to the anterior region of the acrosome in caudal spermatozoa. Freeze-fracture replicas, examined to study changes in organisation of intramembranous particles of the plasma membrane during sperm maturation, reveal distinct changes in their distribution in the acrosome, post-acrosome and spermatozoon tail, especially in the corpus and cauda epididymidis.

Properties and Structure of Aqueous Solutions of Hydroxypropyl‐beta‐Cyclodextrin

AbstractMolecules of hydroxypropyl‐beta‐cyclodextrin (HPBCD) in aqueous solution interact with each other via hydrogen bonds. Especially for concentrations beyond 50% (w/w), a strong increase in viscosity and a considerable decrease of freezable water can be determined. The addition of substances which are able to break the water structure (urea, KSCN) reduces these effects remarkably. The formation of inclusion compounds with urea could not be detected. Endothermic phase transformations confirm the presence of liquid crystalline structures at low temperatures. These liquid crystalline structures are not detectable at room temperatures, but the freeze fracture technique shows micelle‐like associates of a size of about 20 nm. Studies on the complexation of phenolphthalein as well as surface tension measurements of HPBCD solutions indicate that a minimum concentration of HPBC is necessary for the association phenomena.

Effects of diabetes mellitus on structural and functional properties of erythrocyte membranes.

Diabetic patients present alterations in the activity of a number of enzymes of the plasma membrane. The aim of this study was to verify if the modifications of the enzymatic activities in diabetes mellitus are associated with structural alterations of the cellular membrane. By means of the freeze-fracturing technique, we studied the structure of erythrocyte membranes from 15 insulin-dependent diabetic patients (24-43 years) and 15 age-matched healthy subjects (26-47 years). The kinetic properties of the Na+/K(+)-ATPase of the same membranes were also investigated. The Na+/K(+)-ATPase of the erythrocyte plasma membrane shows an uncompetitive inhibition in the diabetic subjects. As for the freeze-fracturing results, the intramembrane particles of the erythrocyte membranes from diabetic patients appear more clustered with respect to those obtained from controls. The uncompetitive inhibition of the enzyme suggests the presence of conformational modifications of the protein. This hypothesis is supported by the freeze-fracture results which indicate that the integral protein constituents of the membrane in diabetes tend to aggregate. Modifications of the interactions between the enzymatic subunits and the membrane lipid environment might be at the basis of the Na+/K(+)-ATPase alteration in diabetes.

Tight junction complexity in the retinal pigment epithelium of the chicken during development

In the avascular retina of birds, the pigment epithelium (RPE) is the main site of the blood-retina barrier. Tight junctions (TJs) connect the pigment epithelial cells and represent the structural substrate of the barrier function. We investigated, by means of the quantitative freeze-fracturing technique, the TJs of the chicken RPE during development and compared them with the TJs of choroid capillary endothelial cells which are known to be fenestrated. The association of TJs with the protoplasmic membrane leaflet (P-face) is more pronounced in the RPE than in the choroid vessels. Between embryonic day 15 (E15) and E19, we observed a significant increase in the TJ complexity in the RPE, but not in the choroid vessels. The increase coincides with the morphological and functional maturation of the chicken retina suggesting that complex P-face-associated TJs in the RPE are necessary for the formation of an effective blood-retina barrier.

A Method for Determining the Proportions of Different Phases in Hydrated Phospholipids by 31P Nuclear Magnetic Resonance (NMR) Spectroscopy

The ability of hydrated lipids to adopt a variety of phases in addition to the bilayer phase is well documented in the literature. Early significant contributions to this research have been made by employing x-ray techniques, freeze-fracture techniques, and 31P NMR.

Role of freeze-fracture technique in tumour pathology

The freeze-fracture technique is unique in its ability to expose extensive face views of the interior of the cell membranes. This feature is particularly useful for studies of events occurring at the plasma membrane, such as exocytosis of secretory granules and of structural components within the membrane such as the intercellular junctions. We have used freeze-fracture preparations in conjunction with conventional thin sections to study the intercellular junctions in a variety of human tumours.

Freeze-fracture evidence for a novel restricting junction at the blood-brain barrier of the cuttlefish Sepia officinalis.

The blood-brain barrier in the cuttlefish Sepia officinalis has been studied with the freeze-fracture technique. Previous thin-section electron microscopy showed that a restricting junction is formed between perivascular glial processes in microvessels and venous vessels, and between pericytes in arterial vessels; the restriction appeared not to be a classical zonula occludens or septate junction. In freeze-fracture replicas from brain optic and vertical lobe, endothelial cells, pericytes and perivascular glia could be recognized by their morphology and relation to the vascular lumen. In microvessels, endothelial and pericyte membranes showed sparse but uniform distribution of P-face intramembranous particles, with no particular particle aggregations. Perivascular glial membranes had a higher density of intramembranous particles but again no particle alignments characteristic of known restricting junctions were seen, although clusterings of intramembranous particles resembling gap junctions were present. In larger venous vessels, the perivascular glial layer showed a multilamellated organization, but again no arrays of intramembranous particles were detected, although this should be a favourable site for visualization of the restricting junctions. The walls of arterial vessels showed collagen deposits and cell processes with apparent intracellular myofilamentous profiles, but no intramembranous junctional particle arrays. It is concluded that the junctional zone observed in thin section electron microscopy is not associated with aligned aggregations of intramembranous particles detectable in freeze-fracture replicas, strengthening the evidence that this is a novel type of restricting junction.

Müller (glial) cells in the retina of urodeles and anurans reveal different morphology by means of freeze-fracturing

Müller (glial) cells of the retina of various species of amphibia (urodeles and anurans) were investigated by means of the freeze-fracture technique. This was done because Müller cells in anamniotes were believed to differ from those in mammals in that they should lack the so-called orthogonal arrays of particles (OAP) which are a characteristic feature of Müller cells in mammalian retina. However, as we could demonstrate previously (Bergvon der Emde and Wolburg, Glia, 2 (1989) 458), fish retinal Müller cells also contain OAP in their membranes suggesting that OAP are a general marker of Müller cells in all vertebrates. As demonstrated in this study, Müller cells of urodeles (Batrachoseps attenuatus and Pleurodeles waltlii) are OAP-positive, whereas two anurans ( Rana esculenta and Xenopus laevis) do not reveal any OAP in their Müller cell membranes. Under phylogenetic aspects, it appears very interesting that frogs are as yet the only vertebrate group that deviates from all other vertebrates in terms of Müller cell membrane morphology.

The statoconial membrane of the guinea pig utricular macula. Scanning electron microscopic investigation combined with the freeze-fracturing technique.

The superstructure of the guinea pig utricular macula was investigated using scanning electron microscopy combined with the freeze-fracturing technique. The statoconial membrane was composed of the otoconial layer, otolithic membrane and subcupular meshwork. The otolithic membrane consisted of closely arranged filaments with beaded appearance and densely packed globular matrix near the otoconial layer. The subcupular meshwork consisted of long branching filaments cross-bridged to one another. The filaments were continuous with those of the otolithic membrane on one side and with the surface of the epithelium on the other, which fills the space between the otolithic membrane and the macular surface. The otolithic membrane would function as a rigid plate and equally distribute the shear force of the otoconial layer to all the ciliary bundles. The subcupular meshwork may play an important role in transmitting the shear strain of the otolithic membrane to all the ciliary bundles and may also exert an additional dampening effect to prevent unwanted vibration.

Structure of the Plasma Membrane of Uterine Epithelial Cells in Blastocyst Attachment: a Review

Changes in the molecular organization of the plasma membrane of uterine epithelial cells during early pregnancy and, in particular, at the attachment period are reviewed. The review focuses on attachment in rodents but other species are also considered. Alterations in protein content and type, as determined with electrophoretic and freeze-fracture techniques, and an increase in tight junction complexity occur in several species. Ultrastructural histochemistry shows that glycocalyx carbohydrates of different species both increase and decrease depending on the type of carbohydrate. Changes in membrane cholesterol content also occur and recent studies showing major reorganization of the actin-containing membrane skeleton are reviewed to show the dynamism of this plasma membrane during the period of uterine receptivity for attachment of the blastocyst.

Crystal aggregates in articular cartilage as observed in the SEM

Scanning electron microscopy, in conjunction with freeze-fracturing and freeze-drying preparation techniques, was used to characterize the morphology and distribution of crystal deposits foundin situ in the articular cartilage of threepost mortem human knee joints. Energy dispersive analysis and X-ray diffraction were used to analyse the chemical composition of the individual crystals. Results included a unique observation of an aggregate of bow-shaped monosodium urate monohydrate crystals within the mid-zone of the articular cartilage, and two types of a large aggregate of calcium pyrophosphate dihydrate crystals distributed throughout the thickness of the cartilage. These results provided evidence of an organized crystal distribution and therefore supported the idea that local growth factors are important.

A freeze-fracture study of the perineurium in galactose neuropathy: morphological changes associated with endoneurial oedema.

Feeding rats with galactose as 40% of their diet results in peripheral nerve oedema related to the intrafascicular accumulation of galactitol and sodium. In this study, associated changes in the perineurium were examined by the freeze-fracture replication technique. Perineurial cells are linked by tight junctions (zonulae occludentes). In normal animals these are made up of anastomosing strands organized in a belt-like arrangement along the margins of continuous cells. The majority of the tight junctions in the galactose-fed animals displayed structural abnormalities. These ranged from slight separation of the strands to fragmentation and dispersal, with looping of isolated strands. Some of the tight junctions contained large dilated compartments within the junctional network. Short lengths of intramembranous particles, probably representing assembly or disassembly of tight junctional strands, were also observed. The membranes of perineurial cells normally possess numerous openings of caveolae. A quantitative assessment showed that the mean density of these caveolae openings was increased in the galactose-fed rats as compared with controls. The alterations in the tight junctions resemble those that have been produced experimentally in epithelia by subjecting them to abnormal osmotic gradients. They also resemble those seen in human diabetic neuropathy in which osmotic disturbances involving the perineurium have been considered to occur. If the alterations involve the inner layers of the perineurium, they are likely to impair its barrier function. The increased number of caveolae openings in galactose neuropathy may represent a reaction to the endoneurial oedema and indicate that the pinocytotic-like vesicles have a transport function.

Zymogen Granules of the Pancreas and the Parotid Gland and Their Role in Cell Secretion

Publisher Summary This chapter highlights zymogen granules (ZGs) of pancreas and the parotid gland. It describes the overall variations of the ZG compartment and the variations of the granule size under a variety of physiological conditions. The chapter also examines the origin and fate of ZGs as observed by cytochemical methods and summarizes the contribution of freeze–fracture techniques to the definition of the ZG membrane architecture. The electrophysiological properties of ZGs and the influence of the cytoskeleton on secretion are discussed. The pancreas and the parotid gland process their macromolecular products by cisternal packaging and exocytosis. The size distribution of the ZGs in the parotid gland cells shows some points of similarity with that observed in the pancreas. During exocytosis the ZG membrane fuses with the luminal plasmalemma, allowing the discharge of granule content. The cytoskeleton controls the movement of ZGs toward the cell apex. The nature of these interactions may be much more complex than a simple ZG alignment in the apical cytoplasm. The life of a ZG starts in the trans-Golgi saccule, in the form of condensing vacuole (CV). Both size and electron opacity of the forming CV are highly variable.

Induction of Milkfat Crystallization in the Emulsified State by High Hydrostatic Pressure

AbstractThe effect of high hydrostatic pressure to milk fat in the emulsified state has been studied by transmission election microscopy (TEM) and low resolution pulsed nuclear magnetic resonance (NMR) for the purpose of following pressure‐induced crystallization processes. Pasteurized dairy creams (35 and 43% fat) were subjected to pressures between 100 and 500 MPa for 1 to 15 minutes at a temperature of 23°C. By applying the freeze fracture technique for TEM studies it was demonstrated that pressurization induces fat crystallization within the emulsion droplets, predominantly at the globule periphery. The ultrastructural findings were confirmed by NMR which showed that the amount of crystallized fat formed depended both on intensity and time of pressurization. In the pressurized cream samples the crystallization proceeded during further storage at 23°C whereas the fat droplets in the original cream sample remained liquid under these conditions, provided that the cream sample was held at this temperature.

Surface layers of Eubacterium yurii subsp. yurii and their possible role in test-tube brush formation and iron acquisition

Eubacterium yurii subsp. yurii is an anaerobic, gram-positive rod. On isolation E. yurii forms cellular arrangements resembling test-tube brushes (TTB). Although TTB decreased in size and number on repeated laboratory subculture in enriched media, media poor in available iron enhanced formation of these. Plasmids were not demonstrated, even after chloramphenicol enhancement. To characterise the nature and possible physiological roles of the structures of the TTB, they were examined by transmission electronmicroscopy (TEM) with thin-section, negative-staining, shadow-casting, freeze-etching and freeze-fracturing techniques, and by scanning electronmicroscopy (SEM). Previous studies by phase-contrast microscopy revealed an amorphous core, the size of which varied in direct proportion to the number of associated bacterial cells. Thin sections of the TTB showed a gram-positive cell wall with additional surface layers. Negative staining, shadow casting and freeze etching revealed a surface layer comprising subunits in tetragonal array (P4 symmetry). Shadow casting showed also that the outermost layer of the cells was composed of fibrillar structures closely associated with but distinct from, the tetragonal layer. The fibrils extended from the cell surface in clumps or strands. The presence of these fibrils was confirmed by the freeze-fracture technique and SEM. Chemical analysis of the core material of the TTB showed it to be low in carbohydrate (0.06%) and protein (0.2%). Energy-dispersive X-ray spectrometry showed that the core was composed mostly of iron.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of diazinon on freeze-fracture images of microvilli of intestinal epithelial cells ofTilapia nilotica

The effect of the organophosphate insecticide, diazinon on the intramembranous particles (IMPs) of the microvilli of the intestinal epithelial cells of Tilapia nilotica fish was studied using freeze-fracture technique. Exposing fish to different repeated concentrations of diazinon (1/2LC50) caused a significant decrease in population density of IMPs in P- and E-faces. IMPs of microvilli found in intestinal epithelial cells are thought to represent many kinds of proteins including enzymes. In the present work, it is suggested that diazinon induced a reduction in enzymatic content of the membrane which was accompanied by a decrease in IMPs density of the microvilli.

Ultrastructural Changes Induced by 12‐O‐Tetradecanoylphorbol 13‐Acetate in Pure Cholinergic Synaptosomes of Torpedo Electric Organ

We have studied the morphological changes induced by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment on pure cholinergic synaptosomes from Torpedo electric organ. These changes were studied by both ultrathin sections and freeze-fracture techniques. We found that after a treatment with TPA, a redistribution of synaptic vesicles inside the nerve endings and exocytotic images could be observed. Also, TPA, under conditions that induced the acetylcholine release, did not change the density of intramembrane particles at the synaptosomal protoplasmic hemimembrane leaflet. Similar results were found when calcium was not present in the extrasynaptosomal medium, and our results suggest that acetylcholine release induced by phorbol ester is probably mediated by exocytosis of synaptic vesicles.