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PubMed1984

Freeze-fracture of 2-cell mouse embryos. A new method for fracture of very small and scarce biological samples.

Membrane morphology and development of intercellular junctions in 2-cell mouse embryos was studied with freeze-fracture electron microscopy. In order to circumvent the dual problem of relative scarcity of samples and their very small size (about 50 micrometers per blastomere), both of which made conventional microtomy-induced freeze-cleavage impractical, a new protocol for handling 2-cell embryos was derived from freeze-fracture procedures established for monolayer cultures. Freshly-collected 2-cell embryos were placed within a 2 mm2 area on plastic Thermonox or Polystyrene coverslips which had been previously coated with 0.002% poly-l-lysine. The embryos were then fixed in 2.5% glutaraldehyde buffered to pH 7.3 with sodium cacodylate and containing 0.01% ruthenium red. The embryos were washed three times in buffer and then glycerinated. The next day, the 2 mm2 area of coverslip containing the embryos (75-100) was cut out with a fine scalpel, inverted and placed on a drop of Elvanol-glycerol mixture and frozen in a slurry of liquid nitrogen-cooled Freon-22. Fracturing was achieved in a Balzers BAF 301 Freeze-Etch Unit by positioning the microtome blade underneath a corner of the inverted coverslip and raising the knife until the coverslip 'popped off'. This procedure left the surface of the Elvanol droplet covered with fractured embryos. The exposed tissue was shadowed with platinum and coated with carbon. This protocol produces large expanses of blastomere plasma membrane as well as cross-fractures of entire blastomeres and 2-cell embryos. The replicas revealed blastomeres endowed with a rich assortment of pleomorphic microvilli.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1980

Thin section and freeze fracture studies of the hypophyseal proximal pars distalis in a teleost (Rhamdia hilarii Val.) during different stages of the reproductive cycle.

Chromophilic cells in the proximal pars distalis of the adenohypophysis of Rhamdia hilarii were studied in thin section and freeze fracture preparations. The gonadotropic cells (GTH-cells) exhibit a diversity of form, the frequency of which can be related to stages (maturation, mature and spent) in the sexual cycle. GTH-cells showing a cytoplasm filled with electron dense polymorphic secretory granules and small rough endoplasmic reticulum (RER) vesicles, have been termed non-vacuolated. During the mature gonadal stage, such cells become increasingly vacuolated. The small RER vesicles become dilated and/or fuse, forming a single enormous cisternum (4--11 micrometer diameter), the contents of which show direct contact with the inner nuclear membrane. These morphological aspects support the idea that Rhamdia hilarii possesses only one GTH-cell type. Evidence from freeze fracture replicas suggests that membrane-associated events precursory to exocytosis take place in regions where the cell and secretory granule membranes are in close apposition. Thin section analysis of secretory granule formation revealed their derivation from the dilated extremities of the inner Golgi saccule which appears to resemble the rigid lamella described in other cells. After detachment of the inner saccule, the immature secretory granules appear to enlarge by microvesicular transport. Freeze fracture and ultrastructural data on the morphology of the cells that presumably synthetise growth hormone are also presented.

Animals↗

PubMed1981

Effects of n-alkanes on the morphology of lipid bilayers. A freeze-fracture and negative stain analysis.

The effect of n-alkanes on the ultrastructure of lipid bilayers has been investigated using freeze-fracture and negative stain electron microscopy. It has been found that the morphology of bilayers containing the long alkane tetradecane is quite different from bilayers containing the short alkane hexane. The smooth fracture faces of gel and liquid crystalline state bilayers are unmodified by tetradecane. However, hexane dramatically alters the hydrophobic bilayer interior, producing large (20 to 50 nm) mounds and depressions in the fracture faces. The fracture steps in these multilayer preparations containing hexane are variable in thickness and often considerably wider than the corresponding fracture steps in multilayers which contain tetradecane or are solvent-free. Alkanes also modify the structure of the P beta' or 'banded' phase of phosphatidylcholine bilayers. The incorporation of tetradecane removes the banded structure from both the bilayer's hydrophilic surface, as viewed by negative staining, and the bilayer's hydrophobic interior, as viewed by the freeze-fracture technique. These results are consistent with X-ray diffraction data which imply that long alkanes are primarily located between adjacent lipid hydrocarbon chains in each monolayer of the bilayer, while short alkanes can partition into the geometric center of the bilayer between apposing monolayers.

Alkanes↗

PubMed1995

A quick-frozen, freeze-fracture and deep-etched study of the cuticle of adult forms of Strongyloides venezuelensis (Nematoda).

The cuticle of adult forms of Strongyloides venezuelensis was studied by routine transmission electron microscopy, conventional freeze-fracture and also using quick-freeze and deep-etch techniques. In routine thin sections the cuticle of S. venezuelensis comprises 7 layers: epicuticle, outer cortical, inner cortical, external medial, internal medial, fibrous and basal. Observation of replicas of specimens fractured across the thickness of the body wall, revealed at the epicuticle an ordered array of particles accompanying the cuticular annulations. At the level of the cortical and medial layers we observed few scattered particles embedded in an amorphous matrix without a particular arrangement. The fibrous layer was represented by several parallel lines of ordered particles of similar size. In tangentially fractured specimens, the epicuticle cleaves readily exposing 2 faces, one exhibiting intramembranous particles without any particular arrangement, immersed in a smooth matrix (P face), and the other showing depressions and very few particles (E face). In replicas of fractures submitted to etching, we observed at the level of the cortical, medial fibrous and basal layers an interconnecting fibrous and globous structure which was organized in a different direction at the fibrous layer. The association of freeze-fracture to deep-etch technique revealed the internal structural organization of the cuticle layers showing details that were not seen before using conventional freeze-fracture technique.

Animals↗

PubMed1976

Tridimensional ultrastructure of freeze-fractured rat kidney cortex (studied in stereo).

The tridimensional ultrastructure of the inner cortex of rat kidney has been studied by observing freeze-fractured tissue in stereo. The complex structure of the tubules, fenestrated capillaries and glomeruli is more readily observed through the irregular fracturing of the tissue that occurs in this technique. The ultrastructure of the brush border and interdigitating membranes of the proximal tubules, the structure of the fenestrated endothelial membranes of the capillaries and the ribbon-like appearance of the filtration space between the foot processes of the glomeruli were especially well depicted in stereo images of freeze-fractured renal tissue.

Animals↗

PubMed1983

Freeze fracture through the cytoskeleton, nucleus and nuclear matrix of lymphocytes studied by scanning electron microscopy.

The technique of delaying fixation until after freeze-fracture and thawing, described in an earlier paper (Haggis & Bond, 1979), has been developed further for study of cells in culture, principally mouse lymphocytes stimulated by concanavalin A. Using a thin layer of cells, a cryoprotectant concentration of either 10% glycerol or dimethylsulphoxide, is sufficient to give good structural preservation after rapid freezing and thawing. Nuclear matrices and Triton-permeabilized cells have been prepared from stimulated lymphocytes for comparative study. Polylysine-coated fibrin support films have been found to provide a convenient means of handling cells and subcellular preparations during freeze fracture, critical point drying and mounting for high-resolution scanning electron microscopy.

Cell Nucleus↗

PubMed1977

Intercellular junctions of antennal gland epithelial cells in the crayfish, Orconectes virilis. A freeze-fracture study.

Labyrinth and nephridial canal cells of the crayfish (Orconectes virilis) antennal gland possess two types of intercellular junctions revealed by freeze-fracture studies. Apical margins of the cells are connected by long septate junctions. In replicas, these junctions consist of many parallel rows of 80--140 A intramembrane particles situated on the PF membrane face (EF and PF fracture faces of Branton et al., 1975). Rows of pits are found on the EF fracture face and are deemed complementary to the rows of particles. Moreover, lateral margins of basal regions of the epithelial cells are attached by many intercellular junctions. These contacts are characterized in thin plastic sections by a narrow dense cytoplasmic plaque located subjacent to the plasma membrane at sites of adjoined cells, and 5 to 12 fine strands of dense material that extend across the intercellular gap between adjoined cells. In freeze-fracture replicas, EF intramembrane faces basal to the region of the plasma membrane containing septate junctions exhibit numerous discoid clusters of particles. The particle aggregates, assumed to represent freeze-cleave images of adhering junctions, range from 900 to 3,700 A in diameter, with individual particles about 185 A in diameter. These junctions appear to connect epithelial cell processes formed by basal infoldings of the plasmalemma, and occur between adiacent cells as well as adiacent processes of a single cell. The discrete aggregates of particles resemble replicated desmosomes (Shienvold and Kelly, 1974) and hemi-desmosomes (Shivers, 1976); therefore, they probably do not constitute a basis for electrical coupling between antennal gland epithelial cells.

Animals↗

PubMed1991

Changes in muscle plasma membranes in young mice with X chromosome-linked muscular dystrophy: a freeze-fracture study.

The structure of the muscle plasma membrane of tibialis anterior muscles of X chromosome-linked muscular dystrophy (mdx) mice was studied by the freeze-fracture technique at 3, 7 and 14 days after birth. The ultrastructural features of the freeze-fracture replicas of the muscle plasma membrane alterations in young mdx mice showed a decrease of orthogonal array, orthogonal array subunit particle and intramembranous particle densities on the protoplasmic face. The results are consistent with the previous studies which have shown that the orthogonal arrays are significantly decreased in number in muscle plasma membranes of adult mdx mice and in those of Duchenne dystrophy. However, the immature mdx mouse membranes at 3 days after birth contained as many orthogonal arrays as controls and did not show a statistically significant decrease (P greater than 0.1 by the Wilcoxon rank-sum test). Moreover, the orthogonal arrays were also numerous in young mdx mouse muscle plasma membranes at 7 and 14 days after birth, although the density was less than that of the control mice (P less than 0.01 by the Wilcoxon rank-sum test). These changes in young mdx mouse plasma membranes may precede the later muscle fibre degeneration in this mouse dystrophy and may provide us with an additional clue to the mechanism why mdx mice scarcely show any disability despite the absence of dystrophyn.

Animals↗

PubMed1983

Freeze-fracture observations of ovulated hamster oocytes with their cumulus cells.

Mature unfertilized oocytes in the cumulus oophorus were examined using the freeze-fracture technique. The P face of the oocyte plasma membrane (oolemma) has randomly distributed intramembranous particles (IMPs) with an average diameter of about 10 nm. No marked difference is found between the intermicrovillous and microvillous regions of the oocyte with respect to the size and distribution of IMPs. The E face of the oolemma has fewer IMPs than the P face. The intermicrovillous region is slightly elevated in some places because of close contact with underlying cortical granules. The oolemma of this elevated region and the membrane of the cortical granule have IMPs which are considerably smaller than those in the remaining areas of the oolemma. The IMP density of the P face of the cortical granule membrane is much lower than that of the E face, possibly indicating a membrane specialization required for fusion of the cortical granule membrane with the overlying oolemma at the time of exocytosis. Cytoplasmic lamellae appear as parallel bands of particles in freeze-fractured specimens. No gap junctions are found between the oocyte and cumulus cells, but residual gap junctions are seen among clustered cumulus cells.

Animals↗

PubMed1993

Differential accumulation of cholesterol in Golgi compartments of normal and Niemann-Pick type C fibroblasts incubated with LDL: a cytochemical freeze-fracture study.

Cholesterol accumulation in the Golgi of normal and Niemann-Pick Type C (NP-C) fibroblasts was shown by freeze-fracture electron microscopy using filipin as a probe for unesterified cholesterol. The specific distribution of cholesterol within individual Golgi compartments could be examined because membrane cholesterol forms complexes with filipin that are visible as membrane deformations (pits and protuberances) in freeze-fracture replicas. The density of filipin-cholesterol deformations, quantitated for cis, medial, and trans Golgi cisternae and trans Golgi vacuoles, was shown to increase in a cis to trans direction. After addition of low density lipoproteins (LDL) to cultured fibroblasts for 24 h, the cholesterol content increased within specific compartments of the Golgi. Normal cells showed an increase in filipin-cholesterol deformations in membranes of cis/medial cisternae and trans Golgi vacuoles, whereas NP-C cells showed only an increase in membranes of trans Golgi cisternae. LDL uptake by cells appears to induce a disparate cholesterol enrichment of Golgi compartments of normal and mutant cells. The ability of cells to process endocytosed cholesterol may in part depend on modulation of cholesterol-enriched membrane transport through the Golgi, a function which appears to be defective in NP-C cells.

Cells, Cultured↗

PubMed1988

Immuno-labelling for freeze-fracture: application to a cell surface attachment antigen.

The cell-surface attachment (CSAT) antigen was investigated by using a technique that combines freeze-fracture with thin-sectioning. Chick heart cells were labelled using colloidal gold secondary antibodies, then fractured and shadowed with platinum vapor. The cells were dehydrated and embedded with their gold labels and replicated surfaces still intact. Sectioning revealed gold particles superimposed with external (E) fracture faces. The gold was definitely not coincident with E face particles and did not appear to be associated with pits. Therefore, it can be concluded that the CSAT glycoproteins is not represented by an E face particle. Whether it may be seen as an E face pit, or is not visible at all in freeze-fracture, is still an open question.

Animals↗

PubMed1990

Image analysis of the chromatin organization in the nuclear domains of freeze fractured hepatocytes and lymphocytes.

The complex organization of the interphase nucleus can be analyzed, by way of thin sectioning and also freeze-fracture. This approach has previously been utilized in association with image analysis to quantitatively describe the organization of isolated rat liver nuclei and nuclear matrices. The main nuclear domains which, in section, present marked differences due to their electron-density, can be identified in replicas with more complex procedures, based on the quantitative evaluation of the number of particles per unit area and mainly by using image analysis. A quantitative analysis of the nuclear substructures has been performed by way of image analysis on in situ nuclei of freeze-fractured cells presenting marked differences in the heterochromatin quantity, such as hepatocytes and lymphocytes. The replicated nuclear particles have been classified according to their diameter and the obtained histograms have been quantitatively evaluated. The nuclear domains, heterochromatin, interchromatin, nucleolus, present characteristic ratios among the three main classes of particles; that is, ribonucleoproteins, solenoid filaments and solenoid fibre aggregates. The typical patterns of the nuclear domains can be further stressed by selecting a single class of particles and by examining its topographic localization. While interchromatin and nucleolar domains present a similar quantitative pattern in hepatocytes and lymphocytes, the heterochromatin of lymphocytes contains a significative higher percentage of solenoid aggregates than that of hepatocytes.

Animals↗

PubMed1982

Structural similarities between a membrane-like surface layer on the cell wall of some microbial cells and the lamellar layer of vesicles formed by short-chain phosphonic acids in water. A freeze-fracture study.

A thin layer on the cell wall of some microbial cells, among them such of medical importance (Aspergillus niger, Trichophyton sp.), is characterized by a mosaic-like rodlet pattern in freeze-fracture electron microscopy. A very similar texture could be found in freeze-fractured bilayer vesicles of n-octan phosphonic acid in presence of 50% glycerol. The formation of this structure in phosphonic acid vesicles is connected with the transformation of the bilayer into the solid crystalline state by cooling. The texture could be suppressed by a higher cooling velocity. In absence of glycerol by the normal quick freezing procedure predominantly intermediate stages were fixed. For the interpretation of the structures revealed a model is proposed, where trigonal subunits stick together, because they are hydrophobic on the one side. These subunits are small crystals with a diameter of 4--7 nm. They are in principle also able to construct a bilayer arrangement directly by a "subassembly-selfassembly" mechanism of selforganization. The membrane-like layer on the cell wall of microbial cells may consist of a crystalline bilayer arrangement of similar substances.

Aspergillus niger↗

PubMed1978

Intramembrane changes occurring during maturation of herpes simplex virus type 1: freeze-fracture study.

During the maturation of two strains of herpes simplex virus type 1 (VR3 and Patton), intramembrane changes were detected with the freeze-fracture technique in the viral envelope and the infected cell plasma membrane, and these changes were compared with data obtained from thin sections. Regardless of the strain, the inner leaflet of the viral envelope of extracellular virions was characterized by a density of intramembrane particles (IMP) three times larger than the host nuclear and plasma membrane. Addition of IMP, which probably represent virus-coded proteins, was detected in the viral envelope only after budding from the nuclear membrane, whereas it occurred during envelopment of capsids at cytoplasmic vacuoles. Fused membranes also showed one of their fracture faces covered with a high density of IMP similar to that of the mature virion envelope. The internal side of the membrane leaflet bearing these numerous particles was always characterized by the presence of an electron-dense material in thin sections. In addition, the plasma membrane of fibroblasts and Vero cells showed strain-specific changes: patches of closely packed IMP were observed with the VR3 strain, whereas ridges almost devoid of IMP characterized the plasmalemma of cells infected with the Patton strain. These intramembrane changes, however, were not observed as early as herpes membrane antigens. Thus, application of the freeze-fracture technique to herpes simplex virus type 1-infected cells revealed striking structural differences between viral and uninfected cell membranes. These differences are probably related to insertion and clustering of virus-coded proteins in the hydrophobic part of the membrane bilayer.

Capsid↗

PubMed1981

The distribution of orthogonal arrays and their relationship to intercellular junctions in neuroglia of the freeze-fractured hypothalamo-neurohypophysial system.

Using freeze-fracture techniques, we have investigated membrane specializations of the glia associated with the hypothalamo-neurohypophysial system of the rat. In the paraventricular (PVN) and supraoptic (SON) nuclei, astrocytes in areas of high neuronal density (i.e., magnocellular regions) display orthogonal arrays of 6--7 nm particles solely near gap junctions, while astrocytes in areas of lower neuronal density (i.e., parvocellular regions), contain additional arrays in membranes not displaying gap junctions. Arrays are especially numerous on astrocytic perivascular end-feet in both nuclei and in the laminations of the pial-glial limitans ventral to the SON. Ependymal cells near the PVN show arrays both on their lateral surfaces (displaying gap junctions) and on their apical surfaces (facing the CSF). Tight junctions are not noted on astrocytes or ependymal cells, but are noted on both the somas and myelin lamellae of oligodendroglia. Both of these latter membranes occasionally contain gap junctions as well; however, orthogonal arrays are never noted on oligodendroglia. The plasma membranes of pituicytes in the neurohypophysis display gap junctions, complex junctions, and tight junctions. Orthogonal arrays are noted near the first two of these, but not near the last. Arrays in the neutral lobe appear most dense on membranes adjacent to subpial or perivascular spaces. Pituicyte membranes containing orthogonal arrays appear infrequently near the neural stalk, increasing towards the distal end of the neural lobe. The distribution of orthogonal arrays in this system, as well as in other systems in which they have been noted, suggests a polarization of membrane activity.

Animals↗

PubMed1975

Intramembrane organization of specialized contacts in the outer plexiform layer of the retina. A freeze-fracture study in monkeys and rabbits.

Freeze-fracture analysis of the neural connections in the outer plexiform layer of the retina of primates (Macaca mulatta and Macaca arctoides) demonstrates a remarkable diversity in the internal structure of the synaptic membranes. In the invaginating synapses of cone pedicles, the plasma membrane of the photoreceptor ending contains an aggregate of A-face particles, a hexagonal array of synaptic vesicle sites, and rows of coated vesicle sites, which are deployed in sequence from apex to base of the synaptic ridge. The horizontal cell dendrites lack vesicle sites and have two aggregates of intramembrane A-face particles, one at the interface with the apex of the synaptic ridge, the other opposite the tip of the invaginating midget bipolar dendrite. Furthermore, the horizontal cell dendrites are interconnected by a novel type of specialized junction, characterized by: (a) enlarged intercellular cleft, bisected by a dense plate and traversed by uniformly spaced crossbars; (b) symmetrical arrays of B-face particles arranged in parallel rows within the junctional membranes; and (c) a layer of dense material on the cytoplasmic surface of the membranes. The plasmalemma of the invaginating midget bipolar dendrite is unspecialized. At the contact region between the basal surface of cone pedicles and the dendrites of the flat midget and diffuse cone bipolar cells, the pedicle membrane has moderately clustered A-face particles, but no vesicle sites, whereas the adjoining membrane of the bipolar dendrites contains an aggregate of B-face particles. The invaginating synapse of rod spherules differs from that of cone pedicles, because the membrane of the axonal endings of the horizontal cells only has an A-face particle aggregate opposite the apex of the synaptic ridge. Specialized junctions between horizontal cell processes, characterized by symmetrical arrays of intramembrane B-face particles, are also present in the neuropil underlying the photoreceptor endings. Small gap junctions connect the processes of the horizontal cells; other gap junctions probably connect the bipolar cell dendrites which make contact with each cone pedicle. Most of the junctional specializations typical of the primate outer plexiform layer are also found in the rabbit retina. The fact that specialized contacts between different types of neurons interacting in the outer plexiform layer have specific arrangements of intramembrane particles strongly suggests that the internal structure of the synaptic membranes is intimately correlated with synaptic function.

Animals↗

PubMed1982

Synaptic vesicle activity in stimulated and unstimulated photoreceptor axons in the housefly. A freeze-fracture study.

Light-stimulated and unstimulated photoreceptor (retinular) axon terminals in the lamina ganglionaris (first optic neuropil) of the housefly are examined using freeze-fracture replication. The presence of numerous, cross-fractured capitate projections permits unmistakable identification of the retinular axon terminal membrane. Regardless of the conditions of illumination, the protoplasmic face (P-face) of the terminal membrane contains numerous bowtie-shaped particle clusters (active zones) which resemble the en face form and disposition of the presynaptic ribbon found in thin sections. Estimates from freeze-fractured material indicate that each retinular axon possesses at least 175 such active zones. In eyes fixed during illumination, active zones are surrounded by many membrane dimples indicative of vesicle fusion sites. Such synaptic vesicle sites are seldom encountered in terminals which are dark-adapted and fixed in the dark. Results from light-adapted eyes placed in the dark following the onset of fixation suggest that endocytosis may occur in the extrasynaptic regions of this inhibitory synapse. P-face particles are uniformly distributed throughout the extrasynaptic regions of unstimulated terminals. Particle density increases in areas peripheral to the active zones in stimulated eyes, particularly within the regions presumed to be undergoing active endocytosis. These structural findings are discussed in the context of the Heuser-Reese model of vesicle exocytosis and recycling.

Animals↗

PubMed1976

Freeze-fracture replication of organized tissue without cryoprotection.

Fresh pieces of rat liver and pancreas were rapidly frozen without prior chemical fixation or cryoprotection, and replicated folloing freeze-fracture. Replicas revealed small peripheral areas free of ice crystals or damage and, within such areas, general ultrastructural morphology was essentially similar to that seen in conventionally processed material. On fracture faces of plasma and nuclear membranes a population of less prominent particles in addition to conventional membrane-associated particles was seen, and smooth areas devoid of particles of any type were seen on some nuclear membranes. These smooth areas did not appear to be similar to smooth areas allegedly arising as artifacts of conventional processing. Tight junctions and gap junctions appeared as they do in cryoprotected specimens. The results provide a base-line for assessing the possible effects of processing steps or agents on the ultrastructure of organized tissues as revealed in freeze-fracture replicas.

Animals↗