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PubMed1984

Preservation of the structural integrity of tight junctions during the freeze-fracture of ciliary epithelium.

It has recently been reported that losses of tight junction material could result from the freeze-fracture process. To verify this assumption, we tried to increase the possibility, if any, of losses of junction material, by inducing an important fragmentation of junctional fibrils by bathing ciliary epithelium in a 0.5M sucrose solution before glutaraldehyde fixation and freeze-fracturing at -160 degrees C. In spite of a significant redistribution of junctional material on both fracture faces, careful examination of complementary replicas and measurements of junction elements and interruption lengths showed that no loss of junctional material occurred in this tissue. The influence of physical parameters (i.e. temperature) on the preservation of the structural integrity of the tight junction during fracturing is now a problem to be considered.

Animals↗

PubMed1998

Crystallographic analysis of freeze-fracture electron micrographs: application to the structure determination of cubic lipid-water phases.

An original approach combining freeze-fracture electron microscopy and quantitative image processing has been developed as an alternative to X-ray analysis. It has been applied to the crystallographic study of different lipid-water cubic phases [bicontinuous or micellar and of type I (oil-in-water) or type II (water-in-oil)] and has enabled significant advances in the study of these phases. Freeze-fracture electron microscopy has revealed that the cubic phases fracture preferentially along a few crystallographic directions which appeared on the images as noisy planar fracture surfaces containing periodic information. The visibility of the corresponding unit cells has been considerably improved by image-filtering techniques based on correlation averaging, allowing a quantitative analysis of the fracture images to be made. This analysis yielded faithful information on the symmetries of the cubic structure (rotation axes and mirror planes) as well as on the structure of the cubic phase itself. Eventually, the different parameters that determine the most favourable fracture pathways within the structures were established. This novel approach constitutes a powerful tool of general interest, complementary to X-ray diffraction, for solving complex ordered macromolecular structures at low resolution.

Crystallography↗

PubMed1998

Electroperturbation of human stratum corneum fine structure by high voltage pulses: a freeze-fracture electron microscopy and differential thermal analysis study.

Application of high voltage pulses (HVP) to the skin has been shown to promote the transdermal drug delivery by a mechanism involving skin electroporation. The aim of this study was to detect potential changes in lipid phase and ultrastructure induced in human stratum corneum by various HVP protocols, using differential thermal analysis and freeze-fracture electron microscopy. Due to the time involved between the moment the electric field is switched off and the analysis, only "secondary" phenomena rather than primary events could be observed. A decrease in enthalpies for the phase transitions observed at 70 degrees C and 85 degrees C was detected by differential thermal analysis after HVP treatment. No changes in transition temperature could be seen. The freeze-fracture electron microscopy study revealed a dramatic perturbation of the lamellar ordering of the intercellular lipid after application of HVP. Most of the planes displayed rough surfaces. The lipid lamellae exhibited rounded off steps or a vanished stepwise order. There was no evidence for perturbation of the corneocytes content. In conclusion, the freeze-fracture electron microscopy and differential thermal analysis studies suggest that HVP application induces a general perturbation of the stratum corneum lipid ultrastructure.

Electric Stimulation↗

PubMed1987

Quantitative freeze-fracture and filipin-binding study of retinal pigment epithelial-cell basal membranes in diabetic rats.

Breakdown of the blood-retinal barrier in diabetes may be related to alterations in the retinal pigment epithelial (RPE) cell layer. Morphological studies suggest increased permeability of diabetic RPE plasma membranes, and proliferation and flattening of the RPE basal infoldings have been observed in diabetic animals. In order to determine whether these phenomena are associated with changes in membrane protein or sterol composition, we have used quantitative electron-microscope freeze-fracture and filipin-binding techniques to study the RPE basal membrane in streptozotocin diabetic and 3-O-methyl glucose control rats. Perfusion-fixed retinas were processed for freeze-fracture and filipin-binding analysis. Filipin, a polyene antibiotic, binds specifically to 3-beta-hydroxy-sterols to produce membrane deformations recognizable by freeze-fracture. These analyses revealed an 11% increase in the density of intramembrane particles within the cytoplasmic (P-face) leaflet in diabetic rats as compared with the controls (P less than 0.01, t test). The increase occurred primarily in 6-9-nm particles, while smaller particles were decreased (P less than 0.001, chi-square test). Filipin binding was the same in both groups. These results suggest that alterations in intrinsic membrane proteins may contribute to permeability and surface area changes in the diabetic RPE but that RPE membrane sterols are not affected by diabetes.

Animals↗

PubMed1996

[The freeze-etching and freeze-fracturing technics and their use for studying cellular membranes in the central nervous system].

In the present review, both subkinds of the method for membrane splitting, namely freeze-etching and freeze-fracturing techniques, are presented. After a brief historical survey, a mechanism of their action is described. Special attention is paid to the consecutive stages in applying these techniques--fixation, fracturing, etching, replication, cleaning and observation. A new idea for a better cleaning of the replicas and an explanation of the different occurrence of intramembranous particles on the membrane fracture faces are proposed. Finally, the application of the freeze-etching and freeze-fracturing techniques to visualize the intramembranous structure of membranes in the central nervous system is outlined.

Animals↗

PubMed1983

[Submicroscopic study of brown adipose tissue in young rats by the freeze-fracturing method].

The authors report the results of their ultrastructural study on the rat brown adipose tissue using the freeze-fracturing techniques. It is well known from transmission electron microscopy that lipid vacuoles of the brown adipose tissue cells are bound by a dense line corresponding to a structure as yet totally unknown. The results of the study using freeze-fracturing technics show that the morphology of lipid vacuoles surface is different from that of the typical unit membrane. The possibility is discussed that the particular aspect of the surface is due to the lypolysis induced by tissue fixation.

Adipose Tissue, Brown↗

PubMed1976

Organization of the nucleoplasm in Escherichia coli visualized by phase-contrast light microscopy, freeze fracturing, and thin sectioning.

The organization of the nucleoplasm in Escherichia coli was studied by comparing the results obtained by freeze fracturing and thin sectioning. In addition to exponentially growing cells, we used chloramphenicol-treated cells which show a well-defined nucleoplasm, in the phase-contrast light microscope and can therefore function as a control for treatments necessary for electron microscopy. Two factors were found to determine the visibility of the nucleoplasm in freeze fractures: first, the state of lateral aggregation of deoxyribonucleic and fibrils, which is enhanced by postfixation with OsO4 according to the Ryter-Kellenberger technique; second, the presence of ice crystals. When their formation is prevented by the use of high concentration of freeze-protecting agents, the nucleoplasm appears as a smooth region in cells that have been prefixed. In unfixed cells, however, the freeze-protecting agent causes disappearance of the nucleoplasm by rearrangement of structures within the cell. This observation makes it hard to determine whether the deoxyribonucleic acid in vivo dispersed, as found after glutaraldehyde prefixation, or compact, as after OsO4 prefixation.

Cell Nucleus↗

PubMed1986

The human parathyroid chief cell--a model for a polypeptide hormone producing endocrine unit as revealed by various functional and pathological conditions. A thin section and freeze-fracture study.

The ultrastructure of the human parathyroid chief cell has been studied by comparing thin section with freeze-fracture findings in close consideration of functional activity. Assessment of function was based on the careful analysis of normal and pathological conditions of the parathyroids in patients with various entities of hyperparathyroidism including clinical data. In contrast to the atrophic glands contralateral to an actively secreting adenoma, stimulated chief cells especially in cases with acute hyperparathyroidism (hypercalcemic crisis), display several remarkable findings: an enlargement of the nucleus with an increase in pores, an abundant amount of rough endoplasmic reticulum, many mitochondria and conspicuous interdigitations of the plasma membrane and an extensively developed Golgi apparatus with budding of numerous vesicular structures and dense lysosomal bodies as well as secretory granules. The fine structure of these alterations was assumed to reflect different states of function in consideration of clinical and laboratory parameters. The process of extrusion of secretory granules and the retrieval of membrane material by endocytosis was particularly investigated by comparison of images obtained by thin sections and freeze-fracture replicas. Our results demonstrate that the human parathyroid chief cell may serve as an ideal model for the study of a polypeptide hormone synthesizing cell with a well known feedback mechanism expressed by the various pathological conditions in patients with the clinical picture of primary and secondary hyperparathyroidism.

Adenoma↗

PubMed2005

Freeze-fracture examination of cultured human trabecular meshwork cells: effect of dexamethasone.

Glucocorticoids can alter the trabecular meshwork in the aqueous outflow pathway of the eye leading to the development of ocular hypertension and glaucoma. Previous studies have shown biochemical and ultrastructural changes in cultured human trabecular meshwork (TM) cells treated with dexamethasone (DEX). In order to assess how the membranes of these treated cells were responding to this glucocorticoid, we compared DEX-treated and control TM cells using the freeze-fracture technique. Human TM cells were grown to confluence on either Millipore HA filter inserts or glass coverslips and then treated for 14 days with or without 0.1 microM dexamethasone. The cell cultures were then aldehyde fixed and prepared for electron microscopy and freeze-fracture. Junctional complexes in control and DEX-treated cells consisted of gap junctions of various shapes and sizes. No tight junctional complexes were apparent in either control or DEX-treated cells. The majority of vesicle fusion sites on the control cells appeared to be randomly distributed and were few in number. In contrast, DEX-treated cells had a significantly greater density of fusion sites, and the majority of the vesicles were aligned into linear arrays. These findings support our previous findings of increased secretory activity in DEX-treated cultured human TM cells. These in vitro model system data appear to correlate with previous in vivo biochemical, ultrastructural and freeze-fracture data.

Adolescent↗

PubMed1986

Freeze fracture study of human corneal endothelial dysfunction.

Intramembrane changes occurring in dysfunctional corneal endothelial cell membranes were examined using freeze-fracture and transmission electron microscopy techniques. Three categories of dysfunctional endothelium were examined: aphakic bullous keratopathy, pseudophakic bullous keratopathy and Fuchs' endothelial dystrophy. Keratoconus corneas and a donor eye bank eye were examined as normal controls. Four intramembrane changes were observed on replicas of freeze-fractured membranes in each category of dysfunctional endothelium. These were a marked reduction in intramembrane particle density on lateral membranes, altered apico-lateral junctional complexes, increased vesicle fusion sites on apical, lateral and basal membranes, and abnormal desmosome-like particle aggregates on the lateral endothelial cell membranes. The marked reduction in intramembrane particles on lateral membranes may be due to a change in the macromolecular components associated with pump dysfunction. The increase in membrane vesicle fusion sites and the breakdown in intercellular junctions may be associated with increased permeability during barrier dysfunction.

Cornea↗

PubMed1984

Lateral diffusion and phase separation in two-dimensional solutions of polymerized butadiene lipid in dimyristoylphosphatidylcholine bilayers. A photobleaching and freeze fracture study.

Mixed vesicles of dimyristoylphosphatidylcholine (DMPC) and a polymerizable lipid containing one diene group per chain are studied by freeze fracture electron microscopy and by the photobleaching (fluorescence recovery after photobleaching) technique. Large thin-walled vesicles of some micron in diameter become more stable after photochemical polymerization. Before polymerization bilayers of the diene lipid exhibit a liquid crystal-to-gel transition at Tg = 31 degrees C. Upon polymerization the transition remains but shifts to a slightly higher temperature (Tg* = 34 degrees C). The transitions in both cases are accompanied by a freezing in of the lateral mobilities. The mixed vesicle exhibits lateral phase separation after polymerization. Before polymerization the two lipids appear miscible at all compositions in the fluid state and at DMPC concentrations at or below 50 mol % in the solid state. After polymerization a two-dimensional solution of the polymer in DMPC is obtained at T greater than Tg*, while lateral phase segregation into DMPC-rich domains and patches of the polymer is observed at T less than Tg*. The domain structure appears identical irrespective of whether polymerization is performed at T greater than Tg or at T less than Tg. A typical value of the diameter of the polymerized lipid domains (approximately 400 A) indicates a rather small aggregation number (N less than 100 monomers). The lateral diffusion coefficient in butadiene-lipid bilayers only decreases from D1 = 3.10(-7) cm2/s to D1 = 8.10(-8) cm2/s (that is by a factor of 4) upon polymerization. This is consistent with the freeze fracture finding of a small aggregation number. We point out the similarities of the mixed vesicles with plasma membranes coupled to the cytoskeleton.

Butadienes↗

PubMed1989

Monolayer freeze-fracture and scanning tunneling microscopy.

This article reviews research on planar monolayer methods, application of the methods to analyses of transmembrane signaling, and the combination of these methods with scanning tunneling microscopy (STM). Past research has involved the development of monolayer freeze-fracture methods. These include monolayer freeze-fracture autoradiography (MONOFARG), an electron microscopic cytochemical method to analyze in-plane distributions of radioisotopes, and double-labeled membrane splitting (DBLAMS) and single-membrane monolayer splitting (SMMS), methods to analyze transmembrane distributions of native and radiolabeled proteins and lipids. Present research has focussed on using these methods to investigate mechanisms of transmembrane signaling mediated by protein kinase C (PKC), including the transbilayer distribution of the tumor promoter TPA, a lipophilic activator of PKC, and the transbilayer distribution of peripheral membrane proteins phosphorylated by PKC. Future work will involve the combination of planar sample preparation with STM. The principles and applications of biological STM are briefly reviewed and a low-resolution STM image of a planar purple-membrane monolayer is included. The combination of planar methods and STM can provide the chemical information lacking in STM images enabling microscopists to investigate biochemical phenomena at nanometer resolution.

Autoradiography↗

PubMed1981

Freeze-fracture study of isolated lysosomes of rat liver.

Freeze-fracture electron microscopy was utilized to pursue the morphological characterization of isolated lysosomes. Combining biochemical data of purity and morphological criteria of identification, it was possible to describe the convex and concave fracture faces of lysosomes. We have compared these with the corresponding faces of tritosomes i.e. lysosomes isolated after injection of Triton WR-1339 into the animal.

Animals↗

PubMed1989

Freeze-fracture of manufactured foods.

Manufacture of food products with desired texture and stability increasingly benefits from an understanding of the food microstructure. Freeze-fracture is advantageous in gaining this understanding because it avoids alterations of hydration, retains fat, and maximally preserves air cells. This paper reviews insightful applications of freeze-fracture to various food crystals, colloids, gels, emulsions, and foams.

Colloids↗

PubMed1982

Rat optic nerve: freeze-fracture studies during development of myelinated axons.

This freeze-fracture study examines the development of myelinated fibers in the rat optic nerve. Axolemma of optic nerve fibers were studied before, during, and after myelination. At birth, the optic nerve is composed entirely of non-myelinated (premyelinated) axons, while in the adult, virtually all fibers acquire compact myelin. Myelination begins at 6-8 days postparturition and proceeds rapidly, such that by 28 days of age approximately 85% of the axons are myelinated. The axolemma of premyelinated fibers from 2-day-old animals exhibits an asymmetrical partitioning of intramembranous particles (IMPs) between E- and P-fracture faces; the E-face had approximately 125 particles/micron2 and the P-face approximately 550 particles/micron2. Particle densities for premyelinated axolemma from 8, 12, 14, 16, and 28-day-old nerves were similar to those observed at 2 days. Beginning at 8-12 days postnatal, definitive association between oligodendroglial processes and axons (termed 'ensheathed' fibers) was observed. At the time of glial ensheathment, there was a 50-100% increase in the number of P-face particles; in contrast, the E-face did not display an overall increase in particle density. In certain regions, however, localized aggregations of E-face particles were observed. IMPs on P-faces of ensheathed axons had a greater mean particle size and higher percentage of 'large' (greater than 9.6 nm) particles than did IMPs on the corresponding fracture face of premyelinated fibers. Myelinated axons from 14-16 day optic nerves displayed several differences from adult myelinated fibers. The P-face of the internodal axolemma had approximately 45% fewer particles than that of adult internodal membrane, and the percentage of large IMPs on the P-face of the younger internodal membrane was approximately 50% of the value for adult internodal axolemma. E-faces of internodal axolemma from 14-16-day-old and adult animals had equivalent IMP densities and size distributions. The nodal region of myelinated axons from 14-16-day-old rats had fewer large particles on both E- and P-faces than did adult fibers, though particle densities on both fracture faces were similar for the two age groups. These studies demonstrate a clear reorganization of axon membrane structure concomitant with axo-glial ensheathment, followed by continued gradual axolemmal changes as myelination progresses.

Aging↗

PubMedDate not supplied

'Shaking pups': a disorder of central myelination in the spaniel dog. IV. Freeze-fracture electron microscopic studies of axons, oligodendrocytes and astrocytes in the spinal cord white matter.

Large axons identified in freeze-fracture replicas of the spinal cord white matter of 'shaking pups' were encircled by spiral processes at the paranodes that appeared to arise from oligodendrocytes. In the axolemma adjacent to some of these processes there were paranode-like intramembranous specializations and node-like accumulations of E-face particles; in other instances, no special contacts formed between axons and the spiral processes. The outer surfaces of the spiral processes, which often apposed astrocytes, frequently contained gap junctions. Although many of the abnormalities identified in this dysmyelinating mutant by freeze-fracture electron microscopy are probably secondary to a more fundamental defect of myelin formation, the prominence of the spiral processes suggests that the encircling of axons by oligodendrocytes may be an independent state of ensheathment and not a passive effect of myelin formation.

Animals↗

PubMed1978

Freeze-fracture replica of the primate lens fibers.

Using freeze-fracture techniques, we studied the fine structure of monkey lens fibers, with special reference to intercellular junctions. Gap junctions were frequently observed at the 'ball-and-socket' interdigitations, the 'tongue-and-groove' interdigitations, and between cortical lens fibers, while tight junctions were never apparent.

Animals↗

PubMed1978

Freeze fracture aspects of the spiral limbus.

Replicas of the freeze fractured spiral limbus are studied in chinchillas. Zonulae occludentes are demonstrated between the interdental cells and the inner sulcus cells; gap junctions are described connecting interdental cells and inner sulcus cells to one another and they are found between the perilymphatic cells in the spiral limbus.

Animals↗