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PubMed1994

Membrane structure of parallel-fibre synaptic terminals in the cerebellum of the jaundiced Gunn rat: freeze-fracture and E-PTA study.

Parallel-fibre synaptic membranes were examined by freeze-fracture and ethanolic-phosphotungstic acid methods in the cerebellum of homozygous (j/j) Gunn rats with hereditary jaundice. Parallel-fibre synapses with dendritic spines of Purkinje cell were severely affected since many Purkinje cells degenerated during the early postnatal period. Some parallel-fibre synaptic terminals lacked their postsynaptic partners and faced astrocytic processes from 18 days of age to the adult stage. These parallel-fibre terminals contained clusters of synaptic vesicles adjacent to synaptic membranes, and synaptic membranes and synaptic cleft materials were identical to those of parallel fibres with postsynaptic partners, as visualized by conventional electron microscopy with osmium tetroxide postfixation and staining of sections with uranyl acetate and lead citrate. In freeze-fractured specimens, the presynaptic membrane of parallel fibres had diffusely distributed large particles and tiny pits on the P-face and protuberances on the E-face, together representing synaptic vesicle attachment sites. Such vesicle attachment sites were present on the presynaptic membranes of parallel fibres without postsynaptic partners from day 18 to the adult stage. After ethanolic-phosphotungstic acid staining, parallel-fibre terminals displayed presynaptic dense projections, intercleft materials and postsynaptic thickening, but some parallel fibres lacked postsynaptic thickening. These observations suggest that the presynaptic membrane structure of the parallel fibre is preserved, even in the absence of a postsynaptic partner, in j/j cerebella. A mechanism for persistence of presynaptic membrane structures without postsynaptic partners in j/j cerebella is discussed.

Animals↗

PubMed1981

Cryofixation of monolayer cell cultures for freeze-fracturing without chemical pre-treatments.

A cryofixation method is presented which gives excellent ultrastructural preservation of monolayer cell cultures without any chemical pretreatments. Rat hepatocytes in primary culture were used in this study. The equipment needed is inexpensive and easy to manufacture. Cells are grown on a usual tissue culture support material (Thermanox plastic sheets). For cryofixation, samples are prepared essentially by a combined sandwich-cryogen-jet technique, 3 mm large discs are punched out and sandwiched with Cu- or Au-object holders of little mass; a 15 micrometer spacer is put in between. The viability of the cells is not impaired by the manipulations before freezing. The sandwich sample is quickly frozen by shooting a propane jet from a simple pressure chamber on to the metal object holder. The relevant parameters were optimized by parallel freeze-fracture analyses of 5% glycerol as a model system and by thermocouple measurements. Sandwich samples are then mounted in an appropriate double replication specimen table for further analysis by freeze-fracturing. It is possible to obtain a certain selectivity of the fracture plane with regard to apical, lateral or basal aspects of the cell layer. Alternatively, disc samples can be processed by chemical fixation methods (including freeze substitution to determine the freeze-fracture plane), since the support material Thermanox is insensitive to organic solvents and easy to cut. In each case the cells remain attached to their substratum throughout the whole procedure. Thus, the ultrastructural data can be directly correlated with parallel functional analyses obtained from the same cell cultures.

Animals↗

PubMed1986

Visualization by freeze fracture, in vitro and in vivo, of the products of fat digestion.

The technique of freeze fracture was used to visualize triglyceride (TG) hydrolysis and the production of lipolytic products (LPs) in vitro and in vivo in the presence of bile salts (BS). Three systems were investigated: pure lipolytic products (oleic acid and monoolein) in the presence of a pure bile salt (taurodeoxycholate (TDC)), lipolytic products produced from TG by pancreatic lipase in the presence of a variety of bile salts, and lipolytic products produced in the intestine of the killifish, Fundulus heteroclitus, after fat feeding. In vitro, lamellae (4-5 nm thick with 0-8-nm water spacings) appeared on the surface of TG droplets in all preparations with LP/BS molar ratios of 1.5 or greater and spherical vesicles (diameter range, 20-130 nm) were produced from these lamellae. With model killifish bile (taurocholate-cholate 1:1) at LP/BS ratios between 1.5 and 4, homogeneous vesicles or particles (mean diameter, 23.8 nm) were produced by lipase at pH 6.9. In vivo, lamellar product phases also occurred after fat feeding. The smallest visible LP/BS structures by freeze fracture electron microscopy were approximately 20 nm globular particles. Large disc-shaped micelles either were not present or were below the resolution limit of the replica (approximately 10 nm). The dominant aggregated lipolytic product phase was composed of multiple layers of rough-textured lamellae. No evidence of cubic structure was seen. These results show that lamellar and vesicular lipolytic product phases can be intermediates in intestinal fat digestion. However, no evidence for the direct endocytotic absorption of these product phases by the intestinal microvillus membrane was found.

Animals↗

PubMed1995

Freeze-fracture study of Bodo sp. (Kinetoplastida: Bodonidae).

Freeze-fracture technique was used to analyse the structure of conventionally fixed and quickly frozen Bodo sp., a free-living kinetoplastid. In the former method, chemically fixed and cryopreserved cells presented a corrugated membrane pattern in the flagella and cell body surfaces. In the latter, however, replicas from quickly frozen unfixed flagellates showed membranes with a smoother aspect, allowing the observation of intramembranous particles (IMPs) on the fracture faces, hardly detectable in previously fixed samples. The IMPs were randomly distributed throughout the cell surface, except in the sparsely seen short IMP rows.

Animals↗

PubMed1978

Fine structure of the palisade zone of the rat median eminence as revealed by freeze-fracturing.

Nerve terminals in the palisade zone of the rat median eminence were investigated with freeze-fracture electron microscopy. Fracture face P of the specific terminals showed two populations of intramembranous particles (IMP): a large and a small variety. The large IMP-s often formed small irregular clusters. In nerve terminals the total number of both populations of IMP-s was considerably less than that found on P membrane faces of ependymal feet. On fracture face E of the nerve terminals, the number of IMP-s was about a quarter of that seen on fracture face P. On both fracture faces of most terminals a few small round impressions (or elevations respectively) were found which may be interpreted as broken necks of either exo- or endocytotic vesicles. Neither gap nor tight junctions occurred at lateral membranes of the specific axon terminals. Similarly, no membrane specializations were observed with freeze-fracturing on membrane areas adjacent to the basement membrane. The findings are discussed in relation to a possible exocytosis mechanism of the hypothalamic releasing and inhibiting hormones.

Animals↗

PubMed1986

"Cholinergic" postsynaptic membranes of bullfrog sympathetic ganglia: electron microscopy of thin sections and freeze-fracture replicas.

"Cholinergic" synapses of the bullfrog sympathetic ganglion cells were investigated with thin sectioning, complementary freeze-fracturing, and deep-etching methods after glutaraldehyde fixation. The protoplasmic (-fracture) face (PF) of the postsynaptic membrane was characterized by intramembranous particles (IMPs), 3,500/micron 2 in density, consisting of larger particles, 10-12 nm in diameter, and smaller ones, 8-9 nm; the complementary exoplasmic (-fracture) face (EF) contained larger and smaller IMPs, 750/micron 2 in density, and numbers of pits. By close inspection of the sections and freeze-fracture replicas at high magnification and with deep-etching in particular, it was concluded that aggregated IMPs might represent transmembranous components and that the particulate entities existing in the postsynaptic active zones might be larger in number than those exposed to view and counted here in the "cholinergic" synapses. An individual IMP often appeared to consist of five or six subunits arranged in a rosette with a central pit. These findings suggest that the aggregated IMPs, particularly the larger ones, may be closely related to the structure of the nicotinic ACh receptor-ion channel complex.

Animals↗

PubMed1977

Freeze-fracture for scanning electron microscopy.

Two different freeze-fracture methods are explored for preparation of biological material for scanning electron microscopy. In the simpler method the tissues are first fixed and dehydrated. They are then frozen and fractured, and after thawing, critical-point dried. This method has already been used in a number of studies of animal tissues (heart, liver, kidney). It is applied here to the examination of plant material (leaf mesophyll cells). In the second method tissues, or cells, are first infiltrated with cryoprotectant (dimethylsulphoxide) then frozen and fractured, and not fixed until after thawing. The fixed tissues are finally dehydrated and critical-point dried. This method also has previously been used in the study of animal tissues, and is applied here to carrot protoplasts, chicken erythrocytes, and leaf mesophyll cells.

Animals↗

PubMed1981

Glomerular podocyte cell junctions in nephrotic syndrome. II. Freeze-fracture studies in human nephrotic syndrome.

In six patients with nephrotic syndrome and in two control subjects freeze-fracture studies of isolated glomeruli obtained by needle biopsies were performed. In the nephrotic patients replicas of freeze-fractured podocytes showed an increased number of cell junctions. These were represented by incomplete occluding junctions and communicating junctions (gap junctions). There was morphologically a similarity between the glomerular podocyte cell junctions in human and experimental nephrotic syndrome (Kühn et al., 1981).

Adolescent↗

PubMed1987

Freeze fracture analysis of muscle plasma membrane in bupivacaine HCl-induced degeneration and regeneration.

We did freeze fracture analysis of bupivacaine HCl-induced muscle fiber damage. The muscle plasma membrane in rat extensor digitorum longus and soleus muscle was analyzed for the density of orthogonal arrays and caveolae at periods of five minutes to 60 days after application of the myotoxin. In degenerating muscle a decrease in the density of caveolae was observed. In regenerating extensor digitorum longus muscle, a decrease in the density of orthogonal arrays and an increase in the density of caveolae was present. The results show that freeze fracture alterations in fast twitch regenerating muscle are similar to those seen in the muscles of patients with Duchenne muscular dystrophy.

Animals↗

PubMed1977

Identification of keratohyalin in freeze-fracture replicas of rat buccal epithelium.

In freeze-fracture replicas of rat cheek epithelium structures representing various forms of keratohyalin granules as seen in stained thin sections have been identified. Discrete spherical granules present in nuclei and cytoplasm are represented in replicas by aggregates of 6 nm particles, and composite granules by similar aggregates associated with a homogeneous component. The observations tend to support the concept of the structural heterogeneity of keratohyalin granules.

Animals↗

PubMed1980

Nodal and paranodal membrane structure in complementary freeze-fracture replicas of amphibian peripheral nerves.

Complementary freeze-fracture replicas of frog peripheral nerves have revealed new details of membrane structures at the node of Ranvier and paranodal axon-Schwann cell junction. At the node both E and P fracture faces of the axolemma have high particle concentrations (approximately 1350/sq. micron and 1600/sq. micron respectively) and these particles do not overlap when tracings from the respective fracture faces are superimposed. A high proportion of the E face particles are large (> 9.5 nm) and cast long shadows while the proportion of large particles in the P face is much lower. In the paranodal region the diagonal pattern of parallel rows in the junctional axolemma always has the same orientation within a given fracture face. In the E face, the parallel rows form a positive (+ 30 degrees) angle to the groove below and in the P face, a negative (-30 degrees) angle to the ridge above. This implies that the diagonal pattern derives from asymmetric subunits that are able to associate along only one axis and are unable to 'flip over' with respect to the junctional membranes.

Animals↗

PubMed1976

Freeze-fracture studies of frog atrial fibres.

The freeze-fracturing technique was used to characterize the junctional devices involved in the electrical coupling of frog atrial fibres. These fibres are connected by a type of junction which can be interpreted as a morphological variant of the "gap junction" or "nexus". The most characteristic features are rows of 9-nm junctional particles forming single or anastomosed circular profiles on the inner membrane face, and corresponding pits on the outer membrane face. Very seldom aggregates consisting of few geometrically disposed 9-nm particles are found. The significance of the junctional structures in the atrial fibres is discussed, with respect to present knowledge about junctional features of gap junctions in various tissues, including embryonic ones.

Animals↗

PubMed1977

Architecture of the outer membrane of Escherichia coli K12. II. Freeze fracture morphology of wild type and mutant strains.

Freeze fracturing electron microscopy of Escherichia coli K12 cells showed that the outer fracture face of the outer membrane is densily occupied with particles. On the inner fracture face of the outer membrane, pits are visible, which are probably complementary to the particles at opposite fracture face. This observation suggests that the particles are micelle-like. In some mutants which lack one or more major outer membrane proteins the density of particles is reduced. The loss of protein d appeared to a prerequisite for this phenomenon. However, mutants which lack all glucose and heptose-bound phosphate in their lipopolysaccharide also have a reduction in particle density whereas, the amount of protein d is normal. Moreover, loss of lipopolysaccharide by EDTA treatment also caused a reduction in the density of particles. From these results it is hypothesized that the particles consist of lipopolysaccharide aggregates stabilized by divalent cations and probably complexed with protein and/or phospholipid.

Bacterial Proteins↗

PubMed1975

Freeze-fracture study of human brain tumors.

This freeze-fracture study was performed in 3 astrocytomas, 6 glioblastomas, 2 ependymomas, 3 medulloblastomas, 1 cerebellar sarcoma, 3 germinomas, and 1 medulloepithelioma. The number of nuclear pores/mum2 nuclear membrane was not correlated with biological malignancy. Fracture faces A and B were discernible in nuclear, Golgi and rough endoplasmic reticulum (ER), mitochondrial surface, and plasma membranes. Fenestrae were evident in Golgi and ER membranes. The transitional zone of cristae from the inner surface membrane appeared as a circular hole and broken-off neck on faces A and B of the inner surface membrane, respectively. The decrease in number of membrane particles in the plasma membrane seemed to correlate with the frequency of metastases, and, in addition, the membrane particles appeared to cluster in glioblastoma, medulloblastoma, and medulloepithelioma. The gap junctions were abundant in astrocytomas, moderate in number in ependymomas and germinomas, and rare in glioblastomas, cerebellar sarcoma, and medulloepithelioma. Tight junctions were often found in germinomas and medulloepithelioma, and rarely in ependymomas.

Astrocytoma↗

PubMed1979

Polycythemia vera: electron microscopy of the bone marrow in 10 non-treated patients. A thin section and freeze-fracture study.

Electron microscopy (thin sections and freeze-fracture replicas) was performed on the bone marrow of ten patients with Polycythemia vera prior to any treatment. In addition to a hyperplasia of all three cell lineages and the sinuses, atypias were observed in the maturing erythroblasts. These aberrations of normal development consisted mainly of deep invaginations of the nuclear envelope in proerythroblasts and conspicuous nuclear clefts in erythro- and normoblasts. In comparison with similar changes in dyserythropoietic and aplastic anemia as well as leukemia these alterations are discussed in connection with disturbances of DNA synthesis. Further atypias involved megakaryopoiesis which displayed microforms probably as an evidence for maturation arrest. These ultrastructural abnormalities and their morphological features of a neoplastic proliferation of all three cell lineages in Polycythemia vera are in good agreement with the new concept of a transformation of a pluripotent stem cell with clonal character.

Adult↗

PubMed1987

Freeze-fracture cytochemistry of rat glomerular capillary tuft. Determination of wheat germ agglutinin binding sites and localization of anionic charges.

We propose here the use of freeze-fracture to gain access and to label in vitro glomerular components and locate WGA receptors and anionic sites. Tissues are frozen, fractured under liquid nitrogen, and thawed. Freeze-fracture rendered all glomerular structures directly accessible to the reagents. This made possible study of the nature and topology of cationized ferritin and WGA binding sites. WGA-gold complexes were observed over plasma membranes of podocytes and of endothelial and mesangial cells. Labeling of podocytes and endothelial cells was similar in the mesangial area and in the peripheral part of the capillary loop. Cross-fractures of extracellular matrices showed that WGA bound uniformly to the glomerular basement membrane (GBM) as well as to mesangial matrix. In fractured specimens treated with neuraminidase, WGA was no longer observed over podocytes but it consistently labeled the surface of endothelial and mesangial cells. Whereas in GBM cross-sections WGA binding was greatly reduced or even abolished, it remained unmodified in the mesangium. This shows that only NeuNAc (sialic acid) might account for the binding of WGA to podocytes, whereas GlcNAcs appear to be the main WGA binding sites on endothelial and mesangial cells and in the mesangial matrix. Both NeuNAc and GLcNAc residues are probably associated in GBM. With cationized ferritin (pI 8.3) at pH 7.4, intense, continuous labeling was seen all over the different plasma membranes, denser in podocytes than in endothelial cells. CF was also observed in cross-fractured profiles of extracellular matrices and never appeared agglutinated in discrete sites.

Animals↗

PubMed1987

Intercellular junctions between fibroblasts in connective tissues of the eye of macaque monkeys. A thin section and freeze fracture analysis.

Thin-section electron microscopy and freeze fracture were used in the analysis of the intercellular junctions between fibroblasts in connective tissues of the eye of Macaca mulatta, M. fascicularis, and M. arctoides. Fibroblasts located in the subconjunctival loose connective tissue, anterior sclera, scleral spur, iris stroma, ciliary body stroma, and posterior choroid were jointed by three kinds of junctions. Gap junctions were of different sizes and frequently composed of a small number of connexons organized in polygonal aggregates or linear arrays. Tight junctions were represented by isolated strands and never composed a continuous belt around the cells. Intermediate junctions were seen in thin sections but did not have any representation in the interior of the plasma membrane. It remains to be established whether, as is the case in other tissues, pathologic conditions of the eye are accompanied by some changes in the morphology and distribution of intercellular junctions between fibroblasts.

Animals↗

PubMed1977

Freeze fracture studies on the annelid septate junction.

Freeze-fractured preparations of septate junctions between epidermal cells of annelids (Lumbricus terrestris and Tubifex spec.) have been investigated. In Lumbricus the protoplasmic face (PF) of the plasma membrane is characterized by variously arranged rows of particles. Apically the rows take an undulating course and often are separated by wide distances. In the basal part of the junction the rows run closely together and more or less in parallel. The diameter of the particles measures 80--120 A, the distance between two particles (centre to centre) is 150--250 A. Additionally striking rows of large particles (long diameter 150--200 A). Are to be observed mainly near the basal part of the junction. In Tubifex both faces of the plasma membrane could be studied in detail. The protoplasmic face (PF) contains rows of distinct individual particles (mean diameter 100--150 A, centre to centre distance approx. 250 A) whereas the particles of the extracellular face (EF, mean diameter 200-250 A) usually form continuous strands in which the individual particles seem to fuse. The density of arrangement of the strands varies considerably. Additionally ladder-shaped membrane structures have been observed in plasma membranes of this species.

Animals↗