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PubMed1989

Freeze-fracture cytochemistry: review of methods.

"Freeze-fracture cytochemistry" encompasses a diversity of recently developed techniques in which freeze-fracture and cytochemistry are combined. Cytochemical labeling may, in principle, be integrated into one of three basic points in the standard freeze-fracture procedure; 1) before the specimen is frozen, 2) after it has been fractured, or 3) after it has been platinum shadowed and/or carbon coated. Visualization of the labeled cellular structures can be achieved by a variety of different methodologies. For example, the markers (usually colloidal gold particles) may be viewed embedded within a replica, or attached to it via fragments of membrane (or other cellular components). Sectioning is a central strategy in a number of techniques, either in combination with or in place of replication. The different combinations of methods that have been devised are not, for the most part, alternative ways of arriving at the same result; each provides quite distinct information about specific classes of membrane component or other structure in the cell. The purpose of this review is to present, within a single article, a systematic survey of the full range of techniques currently available in freeze-fracture cytochemistry. Emphasis is placed on explaining the principles underlying the methods and on illustrating their applications. With the success recently achieved, freeze-fracture cytochemistry has moved from the phase of experimental development to a position in which it may be expected increasingly to make significant contributions across a wide spectrum of problems in cell and membrane biology.

Animals↗
PubMed1982

Understanding the artefact problem in freeze-fracture replication: a review.

Freeze-fracture and freeze-etching techniques do not provide artefact-free images of native in vivo or in vitro cells and tissues. Each preparation stage can produce specific artefacts which must be recognized and understood if these methods are to contribute meaningful information to cell biology, This paper reviews the latest information available on artefacts in freeze-fracture replication (and etching) methods and points to possibilities for avoiding some of them. Different specimens show different sensitivity to artefactual changes and the final images must be interpreted carefully with regard to the multi-event process that has led to their production.

Cryoprotective Agents↗
PubMed1991

Fluorocarbon/lecithin emulsions: identification of EYP-coated fluorocarbon droplets and fluorocarbon-empty vesicles by freeze-fracture electron microscopy.

Freeze-fracture has been used to examine perfluorodecalin/egg yolk phospholipid emulsions (70:8, w/v%) destined to be used as injectable oxygen carriers. The fluorocarbon displays a specific granular aspect upon freeze-fracture which makes it readily recognizable and allows the distinction between two populations of objects on the micrographs: fluorocarbon droplets and water-filled lipid vesicles.

Egg Yolk↗
PubMed1986

Imaging intracellular elemental distribution and ion fluxes in cultured cells using ion microscopy: a freeze-fracture methodology.

A freeze-fracture methodology was standardized for tissue culture cells to study intracellular distribution of diffusible elements with ion microscopy. Chinese hamster ovary (CHO) and normal rat kidney (NRK) cells grown on a silicon substrate were sandwiched using another smooth surface (silicon, glass, mica) in the presence of spacers and fast frozen in liquid nitrogen slush. The sandwich was fractured by prying the two halves apart under liquid nitrogen. This procedure produced large areas on the silicon substrate containing hundreds of cells grouped together and fractured at the apical cell surface. After freeze-drying, these cells revealed a subcellular distribution of Na, K, Ca, Mg, P, Cl and S with the approximately 0.5 micron lateral resolution of the ion microscope. Between the nuclei and the cytoplasm of cells, no major differences were observed for Na, K, Mg, P, Cl and S intensities. Calcium alone, however, exhibited a remarkable distribution. Calcium accumulated more in the cytoplasm than in the nuclei of cells. Even within the cytoplasm its distribution was heterogeneous, suggesting Ca binding sites. The fractured cells consistently exhibited high K-low Na intensities. The injured or dead cells were easily recognized among the healthy ones due to their abnormal ion composition. This simple freeze-fracture methodology allowed fracturing of cells without removing the cells from the substrate. In addition, it eliminated the need for washing the nutrient media away and cryo-sectioning before ion microanalysis. The methodology was successfully extended to 3T3 mouse fibroblast, PtK2 rat kangaroo and L5 rat myoblast cultures.

Animals↗
PubMed1991

Validation of convection-limited cooling of samples for freeze-fracture electron microscopy.

Rapid freezing is the most important step in sample preparation for freeze-fracture and other cryotechniques for electron microscopy. A simple heat transfer model is experimentally validated to show that convection from the cryogen to the specimen is the limiting step in rapid freezing of small samples [Biot modulus, (hd/k) less than 1] by measuring cooling rates in a variety of samples, materials, and cryogens. In comparison to the commonly accepted conduction-limited model, the convection-limited model predicts, and our experiments show, that cooling rates are proportional to the surface area to volume ratio, independent of the sample thermal conductivity, and inversely proportional to the product of sample density and heat capacity. We show that almost any material can be frozen at similar rates if the sample thickness, the cryogen, and the method and velocity of contact with cryogen are similar. Liquid ethane or propane cooled to liquid nitrogen temperature are shown to give the best results.

Ethane↗
PubMed1982

Monolayer freeze-fracture autoradiography: origins and directions.

Monolayer freeze-fracture autoradiography (MONOFARG) is a product of two earlier methods: freeze-fracture autoradiography (FARG) and cell monolayer freeze-fracture. MONOFARG incorporates many of the basic principles and cytochemical goals of FARG while exploiting the technical advantages of monolayer freeze-fracturing. The latter method offers the opportunity to process freeze-dried 'half' membranes at room temperature. Although the feasibility of MONOFARG has been shown for qualitative analyses of split membranes, it's quantitative feasibility for transmembrane and in-plane analyses has just begun to be documented. An example of one aspect of that documentation is included in this report. The distribution of 125I-FITC-Concanavalin-A in the plane of split plasma membranes, human erythrocyte extracellular fracture faces, was examined and found to be homogeneous. The relevance of this finding to recently described double labelled membrane splitting experiments is discussed. The future of MONOFARG appears promising, especially in the application of the technique to biologically significant questions.

Autoradiography↗
PubMed2001

Freeze-fracture studies on lipids and membranes.

Freeze-fracture electron microscopy is especially useful for investigation of lipid structures by the advantageous fracture course within hydrophobic zones. Freezing is, on the other hand, a restriction because the structures of lamellar and non-lamellar phase states with disordered acyl chains (L(alpha), H(II,) cubic) are difficult to preserve. An important aspect of this method is therefore the lipid structure of phase states with ordered acyl chains (crystal, gel), and with a different degree of hydration. Freeze-fracture of pure lipid systems creates a valid representation of the structure of non-lamellar phases and of the general structure of the "lamellar" lipid bilayer, and lamellar phases with characteristic deformations (ripples, curvatures, plane sectors) can be identified. Fracture through the hydrophobic bilayer centre of biological membranes reveals characteristic protein components, the intramembraneous particles (IMPs). The lateral distribution of the IMPs is a helpful marker for fluid and rigid phase states, also without deformation of the lamella. The overall history and the present state of knowledge concerning the different structures revealed by the freeze-fracture and freeze-etch techniques in lipid systems, and to a limited extent in biological membranes, is reviewed, taking into account studies from our own laboratory.

Animals↗
PubMed1991

Gap-junction quantification in biological tissues: freeze-fracture replicas versus thin sections.

The relative efficiency of freeze-fracture replicas versus thin sections for the visualization and quantification of gap junctions in biological tissues has been evaluated. Both methods may underestimate gap-junction number--thin sections for reasons of tissue resolution and freeze-fracture replicas due to the mechanics of the fracturing process. Freeze-fracture misses gap junctions in regions of plasma membrane which are highly contoured, such as the overlapping basal cell processes of Drosophila imaginal wing discs and the interdigitating lateral membrane plications of intercalated discs in cardiac tissue. If the missed gap junctions are relatively large, as they are in both of these examples, freeze-fracture significantly underestimates the total gap-junctional area. Thin sections may miss small gap junctions, but in tissues which contain a range of gap-junction sizes the lost junctions constitute a relatively small fraction of the total junctional area. In neoplastic imaginal wing discs, thin sections were as efficient as freeze-fracture replicas in identifying even the smallest gap junctions. Although freeze-fracture may be the better technique for the qualitative and quantitative documentation of small gap junctions in tissues with relatively flat to gently contoured plasma membranes and thin sections may be the superior method for gap-junction quantification in tissues containing a range of gap-junctional sizes and highly contoured cellular processes, the data suggest that a combination of the two approaches should be utilized whenever possible.

Animals↗
PubMed1975

Ultrastructure of Anaplasma marginale after freeze-fracture.

Stained thin sections and freeze-fractured replicas of Anaplasma marginale-infected bovine erythrocytes were examined by electron microscopy. Freeze-fracture replication not only verified basic Anaplasma ultrastructure, but also allowed visualization of structures not previously reported. Because of the partial 3-dimensional views obtained with freeze-fracture replication, a new structure that appears as a protruding tip was discernible. Also, the surface of Anaplasma's limiting membrane was less granular than the fractured surface of host erythrocyte. A corrugated surface with a periodicity of 10.5 nm was seen when the limiting membrane was fractured.

Anaplasma↗
PubMed2000

Freeze-fracture cytochemistry: a new fracture-labeling method for topological analysis of biomembrane molecules.

Freeze-fracture cytochemistry allows visualization of cellular and molecular characteristics of biomembranes in situ. In this review, we discuss freeze-fracture cytochemistry with special reference to a new cytochemical labeling of replicas, the detergent-digestion fracture-labeling technique. In this procedure, unfixed cells are rapidly-frozen, freeze-fractured, and physically stabilized by evaporated platinum/carbon. The frozen cells are then removed from the freeze-fracture apparatus to thaw and are subsequently treated with detergents. After detergent-digestion, replicas are labeled with cytochemical markers. We demonstrate that the technique is a versatile tool for direct analysis of the macromolecular architecture of biomembranes and allows identification of particular intracellular membrane organelles. In addition, we demonstrate the application of ultrasmall gold to freeze-fracture immunocytochemistry. Freeze-fracture cytochemistry is a valuable technique for investigating topology and dynamics of membrane molecules.

Cell Membrane↗
PubMed1984

[Changes in the orientation of the fracture plane of cell membranes during freeze-fracturing as an index of change in membrane structure].

Using freeze-fracture method it is shown that the direction of the fracture plane is a sensitive indicator of the membrane structural organization. A decrease in the proportion of longitudinal fractures is noted upon the membrane rearrangement presumably associated with changes in the lipid phase, and, on the contrary, an increase in the proportion of these fractures is observed upon some actions resulting, presumably, in changes of the protein phase of membranes.

Candida↗
PubMed1998

Structural analysis of cloned plasma membrane proteins by freeze-fracture electron microscopy.

We have used freeze-fracture electron microscopy to examine the oligomeric structure and molecular asymmetry of integral plasma membrane proteins. Recombinant plasma membrane proteins were functionally expressed in Xenopus laevis oocytes, and the dimensions of their freeze-fracture particles were analyzed. To characterize the freeze-fracture particles, we compared the particle cross-sectional area of proteins with alpha-helical transmembrane domains (opsin, aquaporin 1, and a connexin) with their area obtained from existing maps calculated from two-dimensional crystals. We show that the cross-sectional area of the freeze-fracture particles corresponds to the area of the transmembrane domain of the protein, and that the protein cross-sectional area varies linearly with the number membrane-spanning helices. On average, each helix occupies 1.40 +/- 0.03 nm2. By using this information, we examined members from three classes of plasma membrane proteins: two ion channels, the cystic fibrosis transmembrane conductance regulator and connexin 50 hemi-channel; a water channel, the major intrinsic protein (the aquaporin 0); and a cotransporter, the Na+/glucose cotransporter. Our results suggest that the cystic fibrosis transmembrane conductance regulator is a dimer containing 25 +/- 2 transmembrane helices, connexin 50 is a hexamer containing 24 +/- 3 helices, the major intrinsic protein is a tetramer containing 24 +/- 3 helices, and the Na+/glucose cotransporter is an asymmetrical monomer containing 15 +/- 2 helices.

Animals↗
PubMed1979

Structural states of myelin observed by x-ray diffraction and freeze-fracture electron microscopy.

Coordinated freeze-fracture electron microscopy and x-ray diffraction were used to visualize the morphological relation between compacted and native period membrane arrays in myelinated nerves treated with dimethylsulfoxide (DMSO). Comparison of x-ray diffraction at room temperature and at low temperature was used as a critical measure of the extent of structural preservation. Our x-ray diffraction patterns show that in the presence of cryoprotective agents, it is possible to preserve with only small changes the myelin structure which exists at room temperature. These changes include a slight increase in packing disorder of the membrane, a small, negative thermal expansion of the membrane unit, and some reorganization in the cytoplasmic half of the bilayer. The freeze-fracture electron microscopy clearly demonstrates continuity of compact and native period phases in DMSO-treated myelin. Finally, the use of freezing to trap the transient, intermediate structure during a structural transition in glycerol is demonstrated.

Animals↗
PubMed1990

Freeze-fracture scanning electron microscopy and comparative freeze-etching study of parallel fiber-Purkinje spine synapses of vertebrate cerebellar cortex.

The three dimensional surface morphology, topographical arrangement and intramembrane features of parallel fiber-Purkinje spine synapses of teleost fishes and Swiss albino mice cerebellar cortex have been complementarily studied with the scanning electron microscope (SEM) using the freeze-fracture method and with transmission electron microscope (TEM) by means of freeze-etching replica and thin-sectioning techniques. The SEM fractographs revealed for the first time the outer surface morphology of non-synaptic segments and synaptic varicosities of parallel fibers, which showed clusters of spheroidal synaptic vesicles joined by an amorphous substance. Freeze-etching preparations showed an 'en face' view of P face parallel fiber varicosity (presynaptic P face) characterized by a depressed synaptic active zone with exocytotic vesicle sites, round IMPs (7.5-15 nm), and rod particles (38.5-53 nm in length). Conversely the presynaptic E face exhibited a smooth surface, randomly distributed pits and very low density distribution of round IMPs. The P face of Purkinje spine membrane (postsynaptic P face) showed high density distribution of round or ovoid IMPs, 8-18.5 nm in diameter, and rod IMPs up to 79.2 nm in length. IMPs were also observed beneath the postsynaptic membrane (subsynaptic IMPs) corresponding to the localization of postsynaptic density. The E face of Purkinje spine membrane (postsynaptic E face) showed aggregation of round IMPs, 5.6-17.4 nm in diameter, at the synaptic active zone. Some of these IMPs, particularly the large ones, may morphologically represent the glutamate-receptor ion channels complex. The subsynaptic IMPs may correspond to protein kinases. Granular material and fine filaments were observed in the postsynaptic density. A better insight into the topographical relationship between pre- and postsynaptic structures was obtained of flat and invaginated spine synapses and beside a more realistic view of their glial ensheathment. The SEM fractographs and freeze-etching images were compared with corresponding TEM thin sections for orientation and proper identification of pre- and postsynaptic structures.

Animals↗
PubMed1985

Detection of surface-bound ligands by freeze-fracture autoradiography.

This article describes a new freeze-fracture autoradiographic technique for the detection of radioactive ligands associated with the surface of cells in monolayer or suspension culture. Since freeze-fracture replicas are produced in the conventional way, all membrane features normally seen in freeze-fracture are retained, and autoradiographic grains produced by the labeled ligands are seen superimposed on unaltered exoplasmic membrane fracture faces. To assess the feasibility and resolution of this technique, we compared the surface distribution of alpha 2-macroglobulin and cholera toxin, labeled either with 125I or with colloidal gold, on 3T3-L1 fibroblasts. Both by autoradiography and cytochemical gold labeling, alpha 2-macroglobulin was associated specifically with coated pits, whereas cholera toxin was preferentially found over smaller, apparently non-coated membrane invaginations. Together with data on the surface localization of 125I-transferrin on HL-60 myelomonocytic cells, these results demonstrate the application of this technique for the accurate determination of ligand distribution over large areas of plasma membrane. The simplicity and reproducibility of the method should now allow freeze-fracture autoradiography to become a standard technique for investigating the distribution of both endogenous and exogenous cell surface-associated molecules, as well as the redistribution of such molecules under different experimental conditions.

Animals↗
PubMed1986

Freeze-fracture studies of plasma membranes of astrocytes in freezing lesions.

Epileptic foci are frequently associated with a proliferation and hypertrophy of fibrous astrocytes. Such proliferating astrocytes, which are involved in scar formation and are referred to as reactive, have been characterized by a number of morphological changes involving the cytoplasmic organelles and nucleus. With the development of freeze-fracture techniques, which allow for the splitting and analysis of the macromolecular structure of biological membranes, another morphological characteristic could be used to define reactive astrocytes. This characteristic is the alteration in the normal number and distribution of intramembranous particles. Specifically, there is an increase in the number of orthogonal arrays of intramembranous particles, called assemblies, in the plasma membrane of reactive astrocytes. Besides the presence of assemblies in the plasma membranes of astrocytes, similar particle arrays are also found in the central nervous system (CNS), in cell membranes of ependymal cells, in Müller cells near the inner limiting membrane of the retina, and in satellite cells in spinal ganglia. Assemblies are concentrated in the plasma membranes of astrocytic processes that form the glia limitans at the outer surface of the brain and the perivascular sheath around parenchymal blood vessels in the adult mammalian CNS. Plasma membranes of perineuronal astrocytic processes and astrocytic cell bodies have few, if any, assemblies. The factor responsible for this polarity of assemblies in the normal CNS is unknown. However, this polarity is lost when astrocytes respond to various types of injuries to the CNS. It has been postulated that gliosis, resulting in an epileptic scar, may be a response of cortical astrocytes to changes in the local cellular environment and that this reactivity of astrocytes may be related to plasma membrane changes. However, little is known about the initiation of the astrocytic response or the involvement of the astrocytic plasma membrane. Because of the presence of assemblies, astrocytic plasma membranes are an ideal subject for a morphological investigation of alterations of the cell membrane. Assemblies have a distinctive structure and changes in their number and distribution can be readily identified. Also, since there is polarity of the assemblies in astrocytic plasma membranes, such that their number is greatest in subpial and perivascular areas and least in the perineuronal regions, it is easy to identify when assemblies are added or lost from the astrocytic plasma membranes of a particular brain region.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1982

Effects of amphotericin B and its methyl ester on plasma membranes of Candida albicans and erythrocytes as examined by freeze-fracture electron microscopy.

Freeze-fracture electron microscopy of the plasma membranes of Candida albicans yeast cells and red blood cells treated with amphotericin methyl ester and amphotericin B showed that amphotericin B (50 micrograms ml-1) caused extreme aggregation of intramembranous particles on the protoplasmic fracture face of the C. albicans membrane, and a marked reduction of the density of intramembranous particles. On the other hand, the rearrangement of intramembranous particles induced by amphotericin methyl ester (50 micrograms ml-1) produced elevations of the particle-free membrane domains toward the outside of the cells, so that the particles were aggregated in linear furrows surrounding these elevations on the protoplasmic fracture face, and the corresponding ridges on the exoplasmic fracture face. The density of intramembranous particles was greatly reduced on the protoplasmic fracture face. Both polyenes produced only small changes in the erythrocyte membranes at the same concentration. These results suggest that amphotericin methyl ester affects the ergosterol-containing membranes more than amphotericin B, and that ergosterol has a higher sensitivity for these two polyene antibiotics than cholesterol.

Amphotericin B↗