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PubMed1989

Thin-section and freeze-fracture study of post-mortem changes in dog myocardium.

1. Fragments of dog hearts submitted to 1, 6, 10, 24 and 48 h of autolysis at 20 degrees C were studied with freeze-fracture and thin-section techniques under the transmission electron microscope. 2. The freeze-fracture replicas revealed maximal reduction in the mean number and clustering of intramembrane particles at 6 h post mortem, indicating irreversible cellular damage. However, signs of lethal damage (intramitochondrial amorphous dense bodies) were not observed in thin sections of the same material. 3. The present study indicates that signs of irreversible damage similar to that occurring in in vivo ischemic alterations can be detected earlier by the freeze-fracture technique than by the thin-section technique.

Animals↗

PubMed1983

Fusion of Sendai viruses or subviral envelope components with chicken erythrocytes observed by freeze-fracture electron microscopy.

Four different types of envelope of Sendai virus or subviral components, that is, infectious and non-infectious virions, reassembled envelope particles (REP), and Tween-ether-treated envelope fragments (TE), were studied comparatively for membrane interactions with chicken erythrocytes by freeze-fracture electron microscopy, specifically for membrane alteration by envelope fusion. The freeze-fracture replicas of the attachment of the four envelopes in the cold exhibited a common pattern of impressions with attached envelopes, although the fracture plane traversed from erythrocyte to envelope at the periphery of the contact areas of three of the envelopes but not of TE, where the fracture plane mostly cut only through erythrocyte membranes impressed with TE. The freeze-fracture replicas of the four envelopes reacting with erythrocytes after a short incubation period at 37 C exhibited distinctive features: infectious virions and REP displayed evidence of envelope fusion, but non-infectious virions and TE showed a particular pattern of envelope association without fusion. Our data demonstrate that the pattern specific for envelope fusion is the formation of a continuous membrane from envelope to cell membrane in a cross fracture of an erythrocyte.

Animals↗

PubMed1988

Polarity reversal of inside-out thyroid follicles cultured within collagen gel: structure of the junctions assessed by freeze-fracture and lanthanum permeability.

The organization of tight junctional complexes (TJs) was studied in cultured porcine thyroid cells during the inversion of polarity induced by collagen-embedding of inside-out follicles, using freeze-fracture replicas and lanthanum penetration. During the early steps of polarity reversal, freeze-fractures showed that TJs generally persisted. They increased in width and progressively branched out into the basolateral surfaces, towards the basal pole. Later, the number of TJ strands decreased and gap junctions inserted within TJ networks were found between cells in reversed follicles, in the same manner as in typically polarized follicles, embedded in collagen or in suspension. The de novo formation of TJ complexes was rarely found in the reversing structures. Despite the heterogeneity of TJs assessed by freeze-fracture, impermeability to lanthanum tracer was noted in inside-out structures. During the reversal process, some TJs remained unstained, whereas others displayed permeability to lanthanum. This heterogeneity might be due to the "opening" of a small number of junctions (perhaps only one by aggregate). When the process was achieved after 48 hr in collagen, the tightness of the junctions was complete, confirmed by the absence of lanthanum in luminal cavities of newly formed follicles.

Animals↗

PubMed1987

Node of Ranvier formation along fibres regenerating through silicone tube implants: a freeze-fracture and thin-section electron microscopic study.

Thin-section and freeze-fracture electron microscopy have been used to examine the morphogenesis of the node of Ranvier in peripheral nerves regenerating through silicone tubes. A major question posed by this study is whether node formation in fibres regenerating across a gap recapitulates that occurring in normal development. Node formation occurs concurrently with myelination and follows a similar spatial gradient of progression from a proximal to distal direction along the regenerated nerve. Presumptive nodal sites appear prior to myelin formation and are identified as a prominent subaxolemmal density in thin sections and axonal particle patches in freeze-fracture. Following the appearance of presumptive nodes in regenerating fibres, dimeric particles are inserted into the axolemma adjacent to the node. These particles are in close apposition to the overlying Schwann cell terminal processes and with maturity adopt the same circumferential orientation seen in adult nodes. The nodal axolemma of regenerating fibres shows a characteristic increase in the prominence of its subaxolemmal densification and number of heterogeneously sized particles. Mature regenerated nodes demonstrate a complete annulus of nodal particles indistinguishable from control nodes. The results of the present study show that the nodal architecture of regenerating fibres is a faithful reconstruction of normal mature nodes, thus indicating that the morphological correlates associated with saltatory conduction at the node are present in regenerated nodes.

Animals↗

PubMedDate not supplied

Freeze-fracture electron microscopy of lipid membranes on colloidal polyelectrolyte multilayer coated supports.

Lipid membranes were assembled on polyelectrolyte (PE)-coated colloidal particles. The assembly was studied by means of confocal microscopy, flow cytometry, scanning force microscopy, and freeze-fracture electron microscopy. A homogeneous lipid coverage was established within the limits of optical resolution. Flow cytometry showed that the lipid coverage was uniform. Freeze-fracture electron microscopy revealed that the lipid was adsorbed as a bilayer, which closely followed the surface profile of the polyelectrolyte support. Additional adsorption of polyelectrolyte layers on top of the lipid bilayer introduced inhomogeneities as evident from jumps in the fracture plane. Characteristic lipid multilayers have not been seen with freeze-fracture electron microscopy.

Colloids↗

PubMed1999

Direct immunogold labeling of connexins and aquaporin-4 in freeze-fracture replicas of liver, brain, and spinal cord: factors limiting quantitative analysis.

Direct immunogold labeling and histological mapping of membrane proteins is demonstrated in Lexan-stabilized SDS-washed freeze-fracture replicas of complex tissues. Using rat brain and spinal cord as primary model systems and liver as a "control" tissue to identify preparation and labeling artifacts, we demonstrate the presence of connexin43 in freeze-fractured gap junctions of identified and mapped astrocytes and ependymocytes, and confirm the presence of connexin32 in freeze-fractured gap junctions in liver. In addition, the simultaneous double-labeling of dissimilar proteins (connexin43 and aquaporin-4) is demonstrated in gap junctions and square arrays, respectively, in the plasma membranes of astrocytes and ependymocytes. Finally, double-side shadowing and conventional staining methods are used to reveal the extent of biological material present at the time of labeling and to investigate the dynamics of membrane solubilization, the primary artifacts that occur during labeling, and several factors limiting quantitative analysis.

Animals↗

PubMed1980

Ribosome binding sites visualized on freeze-fractured membranes of the rough endoplasmic reticulum.

Freeze-fracture micrographs of cells of the green alga Micrasterias denticulata stabilized by ultrarapid freezing reveal imprints of polysomes on the rough endoplasmic reticulum membranes. The imprints appear as broad, spiral ridges on the P faces and as corresponding wide grooves on the E faces of the membranes. Distinct 110-A particles with a spacing of 270 +/- 45 A are associated with the P-face ridges. Where imprints of individual ribosomes can be discerned, it is seen that there is a 1:1 relationship between the ribosomes and the 110-A particles, and that the 110-A particles are located in a peripheral position with respect to the polysome spirals. We propose that the 110-A particles could be structural equivalents of ribosome-binding sites, consisting of a molecule each of ribophorins I and II and a nascent polypeptide chain. These observations suggest that the spiral form of polysomes could result from the forces generated by the extrusion of the growing polypeptide chains to one side of the polysome.

Binding Sites↗

PubMed1992

Ordered distribution of membrane-associated dense plaques in intact quail gizzard smooth muscle cells revealed by freeze-fracture following treatment with cholesterol probes.

The surface distribution of membrane-associated dense plaques in intact quail gizzard smooth muscle cells was investigated by freeze-fracture. Replicas of fractured smooth muscle cell plasma membrane showed caveola-free regions with few intramembrane particles, interspersed with caveola-populated areas with a higher intramembrane particle density. Electron microscopy of thin sections of quail gizzard smooth muscle revealed the regions free of caveolae to be occupied by membrane-associated dense plaques; anchoring sites for the contractile filaments of the cell. Demarcation between the caveola-populated and caveola-free regions on the relicated intramembrane surface was not clear and thus provided little information concerning the distribution of dense plaque sites. However, treatment of the smooth muscle tissue with the cholesterol-binding agents filipin or tomatin prior to freeze-fracture allowed the dense plaque sites to be easily observed as the sites remained free of the membrane deformations characteristic of these agents. The dense plaque sites consist of caveola-free oval areas juxtaposed in regular bands that traverse the long axis of the cell. The dense plaque sites on the freeze-fracture replica were confirmed by electron microscopy of thin sections of filipin-treated quail gizzard smooth muscle cells, which showed the plasma membrane associated with the dense plaques to be unaffected by the actions of filipin, whereas that of the caveola-populated region was severely deformed. The observations presented in this study provide evidence for a highly ordered distribution of dense plaques at the cell surface of intact quail gizzard smooth muscle cells and thus corroborate existing evidence for an organized substructure of smooth muscle cells.

Animals↗

PubMed1990

An optimized freeze-fracture replication procedure for human skin.

This in vitro study aimed at substantial modification of the freeze-fracture replication technique (FFRT) which should result in an optimal visualization of the ultrastructure of human skin. The technique was modified in two ways: firstly, the conventional sample holders such as gold cups and copper plates were replaced by silver cylinders (83.5% silver, 16.5% copper) resulting in almost perpendicular cross fractures through the skin. Secondly, the replica cleaning procedure was optimized through the following sequence of treatments. Firstly, a mild tissue destruction was obtained by simultaneous lipid solvation and water extraction with absolute methanol (20 h), followed by protein denaturation with dimethyl sulfoxide (DMSO, 24 h). Subsequently, a final treatment was given using an alkaline sodium hypochlorite solution (20% KOH/13% NaClO; 1:3 v/v, 4 d). After rinsing the replicas for 45 min in aqua bidest, they were mounted on copper grids and examined in the transmission electron microscope (TEM). The combination of the unorthodox fracturing method and the optimized cleaning procedure yielded large, practically undamaged and very clean replicas of near perpendicular cross fractures through human skin. Common handicaps related to current freeze-fracture procedures when applied to skin, such as incomplete cleaning and fragmentation of replicas and oblique or irregular fracturing planes, can largely be avoided in this way. In this paper a complete description of the method is given, and a number of advantages are illustrated with the aid of TEM micrographs.

Freeze Fracturing↗

PubMed1991

Partition of epidermal growth factor receptors on freeze-fractured plasma membranes of A431 cells is affected by the ligand.

The fracture immunolabel technique, which permits assessment of the partition of transmembrane proteins with the inner or outer leaflets of the freeze-fractured membrane, was used to analyze the behavior on fracture of epidermal growth factor (EGF) receptors over the plasma membranes of A431 cells. The receptors partition mainly with the outer leaflet of the freeze-fractured plasma membranes, whereas they become associated with the inner leaflet when they are occupied by the ligand. This modified partition is even more evident after receptor clustering induced by incubation with EGF at 37 degrees C. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) decreases the number of receptors over both inner and outer leaflets. An effect similar to that induced by the ligand is obtained when receptor aggregation is achieved using anti-receptor monoclonal antibodies (MAb). The modified partition therefore indicates receptor activation and appears to be a consequence of receptor cross-linking rather than to reflect a conformational change of the receptor molecule. Parallel immunolabeling with anti-phosphotyrosine antibodies of freeze-fractured EGF-treated A431 cells reveals that the receptors, when activated, are associated only with the inner leaflet of the plasma membrane.

Cell Line↗

PubMed1978

A freeze-fracture study of exocytosis and reflexive gap junctions in human ovarian decidual cells.

Fine-structural features of ovarian decidual cells and their mode of secretion were examined by means of freeze-fracture microscopy. Unique cortical peduncular processes contained secretory vesicles within the expanded peduncle tip, the membrane-leaflets of which exhibited a particle-poor E face adjacent to the vesicle lumen and a P face containing a greater particle number. Exocytosis from attached peduncles involved release of vesicular profiles 40-55 nm in diameter; small particles 8.5-11.5 nm in diameter were also observed at degranulation sites. In fractures revealing the E face of the plasmalemma, cytoplasmic portals at the bases of peduncular stalks were distinguishable from endocytic vesicles. The frequent occurrence of reflexive gap junctions associated with peduncles was shown by freeze-fracture. However, there appeared to be no consistent spatial relationship between gap junctions, secretory peduncles, or sites of exocytosis. Freeze-fracture analysis of the topography of reflexive gap junctional profiles revealed that such gap junctions share basic similarities with intercellular gap jum particle-free aisles. The finding in the present study of reflexive gap junctions occurring between peduncles and the cell soma, as well as between peduncles, suggests that the original definitiof the same cell should be broadened to include any gap junctional specialization formed between portions of the plasma membrane of one cell.

Cell Membrane↗

PubMed1998

Ultrastructure, histological distribution, and freeze-fracture immunocytochemistry of gap junctions in rat brain and spinal cord.

The historical development of concepts of gap junctions as sites for electrical, ionic, and metabolic coupling is reviewed, from the initial discovery of gap junctions linking heart cells, to the current concepts that gap junctions represent 'electrotonic synapses' between neurons. The ultrastructure and immunocytochemistry of gap junctions in heart, brain, and spinal cord of adult rats is examined using conventional thin sections, negative staining, grid-mapped freeze-fracture replicas, and immunogold-labeled freeze-fracture replicas. We review evidence for neuronal gap junctions at 'mixed' (combined electrical and chemical) synapses throughout adult rat spinal cord. We also show immunogold labeling of connexin43 in astrocyte and ependymocyte gap junctions and of connexin32 in oligodendrocyte gap junctions. Ultrastructural and freeze-fracture immunocytochemical methods have provided for definitive determination of the number, size, histological distribution, and connexin composition of gap junctions between neurons in all regions of the central nervous systems of vertebrate species.

Animals↗

PubMed1977

Freeze-fracturing of monolayers (capillary layers) of cell, membranes and viruses: some technical considerations.

A novel hinged device for freeze-fracturing of cell monolayer in the Balzers freeze-etch unit is described. It is economical on biological material and enables oriented adsorption of sheet-like membrane fragments. For freeze-fracturing 'by hand' a monolayer is formed on a positively charged piecie of mica (with polylysine) and this is covered with another piece of mica, thin brass plate of filter paper. Such a sandwich is frozen in liquid nitrogen and fractured by means of forceps. Several modifications of this technique as well as practical examples are described. Among possible application are: negative staining of intramembranous protein particles; chemical or physical analyses of single membrane leaflets; identification of protein complexes by immunoelectron microscopy, etc.

Adenoviridae↗

PubMedDate not supplied

The olfactory and respiratory epithelium in rhesus and squirrel monkeys studied with freeze-fracture technique.

Olfactory and respiratory epithelia was prepared for thin-section and freeze-fracture replicas, and were investigated using electron microscopy. Three cell types were identified in the olfactory epithelium: olfactory receptor neurons (ORN), sustentacular cells (SC) and basal cells (BC); and respiratory epithelial cells (RC) in the surrounding mucosa. ORN and RC were provided with kinocilia. The cilia displayed their characteristic 5-7 rows of proximally situated 'necklaces', seen in freeze-fracture replicas. ORN and SC as well as RC were connected with tight junctions classified as being 'tight', whereas the RC displayed intermediate junctions. No nexuses (gap junctions) were found. It is concluded that the monkey olfactory/respiratory epithelium exhibit the same characteristics as seen in other vertebrates when using thin section/freeze-fracture technique.

Animals↗

PubMed1981

Freeze-fracture architecture and polypeptide composition of thylakoid membranes from euploid Ricinus cells.

The freeze-fracture architecture and polypeptide composition of thylakoid membranes of euploid cells of Ricinus communis L. were examined. Electron microscopic examination of the chloroplasts of 1N 2N and 4N cells revealed little variation in the size of chloroplasts, lamellar structure and internal organization of plastids, despite increases in plastid numbers per cell observed to accompany the increase in nuclear ploidy. Thylakoid membranes from euploid cells were also similar in their freeze-fracture morphology. Two basic types of intramembranous particles were observed on the fracture faces of thylakoid membranes of euploid cells. The endoplasmic fracture (EF) face of experimentally unstacked thylakoid membranes of 1N, 2N and 4N cells contain 2 size categories of particles (115-121 A and 164-166 A), whereas the protoplasmic fracture (PF) face of these membranes contain a single size category of particles (85-88 A). The distribution and size of the EF- and PF-face particles were found to be similar among membranes from cells of the 3 ploidy levels. Analysis of the polypeptide composition of thylakoid membranes from 1N, 2N and 4N cells revealed no difference in the relative proportion of the constituent polypeptides of these membranes. The possible factors involved in the regulation of the development of thylakoid structure and composition in the presence of altered nuclear genome size are discussed.

Ricinus communis↗

PubMed1982

Fine structure of the testis in the toad (Bufo arenarum hensel): a freeze-fracture study.

The present work reports novel findings in the toad testis using freeze-fracture techniques. Tight and gap junctions are observed in the nonfenestrated endothelial cells. Numerous gap junctions are present between interstitial cells. Sertoli-Sertoli junctional specializations in the toad are similar to those described in mammals although they appear to be less elaborate. The appearance of nuclear pores, in the seminiferous epithelium, ranges from a uniformly and apparently random distribution in Sertoli cells to large aggregations of closely spaced pores separated by free areas in the nuclear envelope of spermatocytes. Inasmuch as these features have also been described in mammals, the fact that they are present in amphibia might indicate that they represent a widespread pattern. Concomitantly with increasing chromatin condensation, nuclear pores diminish in round spermatids and seem to disappear in the more mature germ cells. The en face freeze-fracture views of annulate lamellae in the postnuclear cytoplasm and membranous features of the acrosome formation, flagellum, and undulating membrane of elongating spermatids are also described.

Animals↗

PubMed1979

A correlated thin-section and freeze-fracture analysis of guinea pig adrenocortical cells.

Comparison of the fine structural features of guinea pig adrenocortical cells as seen in thin sections with those revealed by freeze-fracture confirms the structural appearance of steroid-secreting cells as interpreted from thin sections and reveals significant new features of the membranous organelles. Smooth-surfaced endoplasmic reticulum appears as a network of tubules, interwoven or in parallel, and as cisternae, fenestrated and non-fenestrated. These elements are tightly packed in the deeper cortical cells, excluding other organelles from their domain. Tubules and fenestrated cisternae possess randomly distributed intramembranous particles on their PF faces, while closely packed non-fenestrated cisternae possess aggregates of particles interspersed with aparticulate regions on their PF faces. These differences in particle distribution suggest functional specialization among the various forms of reticulum. Mitochondria appear as elongated structures of varying shape. Freeze-fracture reveals that all their cristae have circular origins from the inner membrane. Sinuous tubules, which appear as tubules in section, and straight tubules, which appear as lamellae in section, arise from single sites. Flattened sac-like cristae may have multiple circular origins. Definite contact points seen between inner and outer membranes may facilitate passage of molecules, including steroids, into the mitochondrial compartments. Lysosomes and peroxisomes, which are easily identified in thin sections with the aid of cytochemistry, are difficult to identify with certainty by freeze-fracture. Single membrane-bound granules of slightly smaller diameter than mitochondria may represent lysosomes. Smaller granules interconnected with the tubular reticulum, as well as dilated regions of this organelle, may represent peroxisomes. Plasma membranes show no indication of tight junctions but do have abundant gap junctions which show a zonal differentiation: small gap junctions throughout the cortex, medium-sized regularly shaped gap junctions in zona fasciculata externa, and large irregular gap junctions in zona fasciculata interna and zona reticularis. The large junctions cover planar areas as well as surfaces of projections of one cell into another. Such junctions may allow passage of ions as well as of low-molecular-weight substances between the cells, facilitating or even amplifying the response to trophic hormone stimulation.

Adrenal Cortex↗

PubMed1982

Cell surface specializations and intercellular junctions in human amniotic epithelium: an electron microscopic and freeze-fracture study.

Cell surface specializations and intercellular junctions of human term amniotic epithelium were examined by conventional thin-section electron microscopy, after staining with the cationic probes ruthenium red and cationic ferritin, and by freeze-fracture methods. Desmosomes were the predominant type of intercellular junction and often the most apical of the junctional types. In freeze-fracture replicas, desmosomes were characterized by roughly circular areas of large, often irregular, P-face intramembranous particles. Gap junctions were identified in the laterobasal regions between cells. In thin sections they were characterized by a narrow intercellular space, and in freeze-fracture replicas had a typical plaquelike arrangement of P-face intramembranous particles and E-face depressions. Hemidesmosomes at the basal cell surface were characterized by occasional large particles and clusters of particles on both the E and P fracture faces. No evidence of tight junctions was found. The apical cell surface was heavily stained by both ruthenium red and cationic ferritin, indicating the negatively charged nature of this surface. Ruthenium red penetrated between the epithelial cells and bound to anionic materials on the lateral cell surfaces, especially at the location of desmosomes. Below the base of the intercellular cleft, large ruthenium red-positive granules were present in the extracellular matrix. The possibility that the anionic substances in the intercellular region may contribute to the control of permeability in the amniotic epithelium is discussed.

Amnion↗