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PubMedDate not supplied

Host-pathogen interactions in experimental Mycoplasma pneumoniae disease studied by the freeze-fracture technique.

The application of freeze-fracture and organ culture technology to the study of disease caused by Mycoplasma pneumoniae provided a new perspective on host-pathogen interactions. Structural variations of the M. pneumoniae membrane and alterations of the host cell membrane that are induced by this pathogen during experimental infection were observed. Study of freeze-fracture preparations of M. pneumoniae revealed the presence of numerous membrane-associated particles embedded in the inner aspect of the bimolecular leaflet. However, areas of the fractured membrane of cells sometimes had particle-free zones and/or blebs. These particle-free areas were sometimes closely aligned to host tracheal epithelium in organ culture. In addition, marked changes in the membrane integrity of the host epithelium during experimental infection were observed. Deterioration of luminal surface membranes and alterations in the distribution of ciliary intramembranous particles were frequently noted. Ciliary dysfunction was also suggested by observations of abnormally aligned and clumped adjacent cilia. These findings correlated well with biochemical data demonstrating altered host macromolecular synthesis and observations of ciliostasis during experimental M. pneumoniae infection.

Animals↗

PubMed1992

Freeze-fracture studies of salivary glands.

Freeze-fracture electron microscopy was employed to study the general morphology, membrane fusion during exocytosis, intercellular junctions in adult and developing salivary glands, and morphological changes of intercellular junctions in secretory stimulation and neoplastic disorders. Intramembranous particles distributed on plasma membranes and organelle-membranes were determined on the basis of particle density per unit. Relationship between distribution and density of particles and functions of membranes and organelles was discussed. Fused membranes took the form of smooth areas of intramembranous particles free on the PF face in the submandibular acinar cells induced by isoproterenol stimulation. The sequence of membrane fusion during secretion was discussed from these freeze-fracture observations. Tight junctions which limited solute diffusion between adjacent cells formed continuous belts around the juxtaluminal region of plasma membrane. Their permeability function was regulated by the number and the geometrical pattern of the tight-junctional strands. The junction appeared late during gestational life, and its development and morphology correlated with the establishment of electrochemical tightness. Similarity in the location and morphology between developmental and neoplastic conditions might indicate reflect the function and degree of cytodifferentiation in salivary gland. Gap junctions were detected as clusters of particles, representing voltage-regulated intercellular channels connecting adjacent cells. The junctions were developmentally regulated, and their size and frequency correlated with the functional activity and with cytodifferentiation in neoplastic disorders. Desmosomes, which appeared as round or elongated clusters of particles, provided the mechanical stability necessary to maintain the organization of acini and ducts.

Animals↗

PubMed1991

Intramacrophagic Mycobacterium avium bacilli are coated by a multiple lamellar structure: freeze fracture analysis of infected mouse liver.

We used freeze fracture electron microscopy to study the fine structure of Mycobacterium avium inside phagosomes of murine macrophages. M. avium-susceptible C57BL/6 mice were infected with M. avium by intraperitoneal inoculation of 10(8) viable bacilli. We studied the microanatomy of the mycobacteria in 3-month infections of mice, a situation in which bacillary multiplication is extensive. In these samples, freeze fracture revealed that intraphagosomal bacilli were surrounded by a multilamellar coat that was apposed to the cell wall. In thin sections, in contrast, the area corresponding to the coat showed no substructure and was electron transparent (the so-called electron-transparent zone that has been previously reported by others). The multiple lamellae resembled an onionlike assembly that was inserted in between the mycobacterial wall outer surface and the phagosomal membrane. Each lamella of the M. avium coat was made up of parallel straight fibrils with a width of 5 nm. A variable number of lamellae, sometimes up to 10 or more elements, coated individual bacilli. The multilamellar coat was absent around both extracellular M. avium and intramacrophagic M. avium after short-term (45-min) inoculation of mice. The supramolecular organization of the M. avium lamellar coat as viewed here by freeze fracture is similar to that of purified mycoside C (P. Draper, J. Gen. Microbiol. 83:431-433, 1974; K.-S. Kim, M.R.J. Salton, and L. Barksdale, J. Bacteriol. 125:739-743, 1976), a mycobacterial component currently known as glycopeptidolipid (W.W. Barrow and P.J. Brennan, J. Bacteriol. 150:381-384, 1982). We conclude that M. avium bacilli growing in macrophages are surrounded by multilamellar capsulelike structures that contain glycopeptidolipid molecules.

Animals↗

PubMed1977

Freeze-fracturing of dispersions.

In the last few years freeze-fracturing has proved to be a useful method for the investigation of dispersions. The freeze-fracture technique provides, above all, information on the arrangement of the colloidal particles in the liquid medium (e.g. on the state of agglomeration). Furthermore, it leads to conclusions about the structure, shape and size of the colloidal particles. The principles and applications particularly of the so-called drop-freeze-fracture technique are briefly described.

Crystallization↗

PubMed1981

Interpretation of morphological data obtained by freeze-fracturing at very low temperatures.

In freeze-fracture replication the greatest variety of methods exists for the fracturing process itself. The present stage of knowledge allows to evaluate fracture faces obtained by freeze-cleaving at temperatures down to 4 K. Under condensation free conditions the possibility for artefact formation will be restricted to the fracturing and replication process. Experiments on model systems and complex biological systems show that many fine structural details seen on freeze-fractured replicas have to be interpreted as the result of a multi-event process involving plastic deformation, elastic recontraction, collapse phenomena and thermal load induced alterations during replica formation. With some structures the degree of deformation may be reduced by chemical fixation procedures.

Cell Membrane↗

PubMed1987

Freeze-fracture label of functional and dysfunctional human corneal endothelium.

Previous freeze-fracture results from our laboratory have shown a reduction in a population of intramembrane particles in the lateral endothelial membranes from dysfunctional human corneas. The size range of these intramembrane particles corresponds to that which has previously been reported for the glycoprotein enzyme Na, K-ATPase in enzyme enriched freeze-fractured membranes. In order to investigate glycoconjugate changes potentially related to the particle reduction, wheat germ agglutinin (WGA), which has been shown to bind to the sugar residues in the ATPase subunit, was used to label three types of corneas with dysfunctional endothelial cells (Fuchs' endothelial dystrophy, aphakic and pseudophakic bullous keratopathy) and two types of corneas (eye bank and keratoconus) with functional endothelium using the technique of thin section freeze-fracture label. Apical WGA labelling on all types of dysfunctional cells was shown to be drastically reduced in comparison to both types of functional corneal endothelial cells. Lateral membranes of dysfunctional cells, exposed by freeze-fracturing, also showed a great reduction in WGA labelling as compared to the fractured lateral membranes of functional cells. The differences observed in lectin labelling of lateral membranes may be related in part to the decreased intramembrane particle density observed in dysfunctional human corneal endothelial cells.

Cornea↗

PubMed1974

The structure of the zonula occludens. A single fibril model based on freeze-fracture.

Replicas of freeze-fractured toad urinary bladder and gallbladder were analysed in an attempt to determine the fracturing properties and structure of the zonula occludens (tight junction). Chalcroft and Bullivant have proposed that the junction has a double set of fibrils with one set associated with each of the adjacent cell membranes. However, the fracturing pattern that is observed might also result from only a single set of fibrils which is shared by the adjacent membranes if fracturing occurred around either side of the fibrils. These two models predict quite different structures at regions of the junction where tranl sitions are made between face A and face B. The relative heights of face A and face B and the shape of the transition from face A to face B do not agree with that expected according to the two fibril model but agree exactly with that expected if only a single set of fibrils existed. Further evidence for the single fibril model is derived from fractures of the mucosa membrane which cross the junction to the membrane of the adjacent cell without deflection. Such fractures reveal a single ridge which appears to be identical to the juxtaluminal fibril of the junction. In addition, small ridges are occasionally found in place of the grooves on face B which, although not consistent with the double fibril model, is expected if the single fibril model were correct. Although alternative explanations might account for these observations, we believe that the simplest and most consistent explanation is that the zonula occludens fractures as would be expected of a single set of fibrils shared by adjacent cells.

Animals↗

PubMed1985

A modified cleansing procedure to obtain large freeze-fracture replicas.

A method for cleaning freeze-fracture replicas is elaborated which avoids their rolling up and fragmentating during successive steps. Immediately after freeze-fracturing, the replicated tissue is slowly thawed from 77 K to room temperature on solid methanol. Safe passage through the subsequent cleaning steps is facilitated by using solutions of gradually increasing surface tension. After at least 24 h of fixation in methanol, the tissue is digested in half-strength bleach containing 5% ethanol. Following further cleaning in full-strength bleach, the remaining organic material attached to the replica is dissolved overnight at room temperature in 50% saturated sodium hydroxide.

Animals↗

PubMed1991

Freeze-fracture cytochemistry: a simplified guide and update on developments.

A wide variety of methods by which cytochemistry and freeze-fracture can be successfully combined have recently become available. All these techniques are designed to provide information on the chemical nature of structural components revealed by freeze-fracture, but differ in how this is achieved, in precisely what type of information is obtained, and in which types of specimen can be studied. Colloidal gold labelling is the most widely used cytochemical technique in freeze-fracture cytochemistry, and for many of the methods it is indispensable. In principle, there are four points in which the cytochemical labelling step may be integrated into the standard freeze-fracture procedure: (i) before the specimen has been frozen, (ii) after it has been fractured and thawed, (iii) after platinum shadowing or (iv) after completion of the full replication sequence. Retention of the gold label so that it can be viewed with replicas can be achieved by depositing platinum and/or carbon upon the labelled surface, thereby partially entrapping the marker particles within the replica, or by retaining, attached to the replica, fragments of fractured membrane (or other cellular components) that would normally have been lost during the replica cleaning step. Another approach to visualizing the label is to use sections, either with portions of a replica included face-on, or for examining the fracture path through the sample (without replica). Recent developments have centered on the use of replicas to stabilize half-membrane leaflets; not only may these and associated attached components be retained for labelling just before mounting, but they provide a means for manipulating the specimen--specifically, turning it over during processing--so that additional structural information can be obtained. This article aims to explain how modern freeze-fracture cytochemistry works, and how the various techniques differ in what they can tell us about membranes and other cellular structures. With the effectiveness of many of the techniques now demonstrated, freeze-fracture cytochemistry is firmly established, alongside a range of related labelling techniques, for increasing application in cell and membrane biology in the 1990s.

Animals↗

PubMed1994

Surface distribution and partition during freeze-fracture of CD8 antigens on human lymphocytes and on epithelial transfected cells.

Freeze-fracture immunocytochemistry was used to analyse the surface distribution, redistribution induced by antibodies, and partition during freeze-fracture, of CD8 molecules on human T lymphocytes and rat epithelial transfected (FRT-U10) cells. Immunogold labelling of CD8 antigens was uniform over the unfractured cell surfaces of both lymphocytes and epithelial transfected cells. After freeze-fracture, the gold particles were associated with the exoplasmic outer leaflets of the plasma membranes in both cell types. In lymphocytes, incubation with antibodies at 37 degrees C up to 20 min induced patching and capping of the antigens on the unfractured cell surface. After fracture, the patched molecules appeared associated with the protoplasmic inner leaflet of the plasma membranes. Parallel antibody-treatment at 37 degrees C of FRT-U10 cells induced clustering of CD8 molecules but failed to cause further aggregation in larger patches or in caps. After freeze-fracture, the immunolabelling was clustered, but associated with the exoplasmic outer leaflet of the plasma membranes as in untreated cells. The different redistribution induced by antibodies and the different behaviour on fracture of the redistributed molecules in the two cell types may be regulated by CD8 interaction with the cytoskeleton.

Animals↗

PubMed1982

Monolayer freeze-fracture autoradiography: quantitative analysis of the transmembrane distribution of radioiodinated concanavalin A.

The technique of monolayer freeze-fracture autoradiography (MONOFARG) has been developed and the principles, quantitation, and application of the method are described. Cell monolayers attached to polylysine-treated glass were freeze-fractured, shadowed, and coated with dry, Parlodion-supported Ilford L4 photographic emulsion at room temperature. Quantitative aspects of MONOFARG were examined using radioiodinated test systems. Background was routinely less than 2.5 X 10(-4) grains/microns 2/day, the highest overall efficiency was between 25% and 45%, and grain density and efficiency were dependent on radiation dose for iodine-125 and D-19 development. Corrected grain densities were linearly proportional to iodine-125 concentration. The method was applied to an examination of the transmembrane distribution of radioiodinated and fluoresceinated concanavalin A (125I-FITC-Con-A). Human erythrocytes were labeled, column-purified, freeze-dried or freeze-fractured, autoradiographed, and examined by electron microscopy. The number of silver grains per square micrometer of unsplit single membrane was essentially identical to that of split extracellular membrane "halves." These data demonstrate that 125I-FITC-Con-A partitions exclusively with the extracellular "half" of the membrane upon freeze-fracturing and can be used as a quantitative marker for the fraction of extracellular split membrane "halves." This method should be able to provide new information about certain transmembrane properties of biological membrane molecules and probes, as well as about the process of freeze-fracture per se.

Autoradiography↗

PubMed1973

The Chediak-Higashi syndrome. Fine structure of giant inclusions in freeze-fractured neutrophils.

The technic of freeze-fracture and etching has been used in the present study to examine the fine structure of giant inclusions in circulating leukocytes from a patient with the Chediak-Higashi syndrome (CHS). The surface granularity of the membranes enclosing the giant inclusions differed slightly from that of normal sized organelles. Two types of giant granules were distinguished in replicas of freeze-fractured CHS neutrophils. The difference in fine structure suggests that one variety is a massively enlarged, but essentially unaltered primary lysosome, while the other develops as a result of continued fusion of small organelles with huge inclusions throughout the stages of polymorphonuclear leukocyte maturation.

Cell Membrane↗

PubMed1979

Three-dimensional view of the chromatin in freeze-fractured chicken erythrocyte nuclei.

The freeze-fracture thaw-fix (FfTF) technique described in earlier papers is applied in the present work to more detailed study of the chicken erythrocyte, by transmission replicas and high resolution scanning electron microscopy (3 nm scan beam size). The three-dimensional structure of the chromatin, and possibly the non-histone protein matrix, of fractured nuclei is to a large extent retained in this method of preparation and seen in stereomicrographs. In these micrographs the helical sub-structure of the 25 nm chromatin strands can be seen at about the same resolution as that of previously published micrographs in which extracted chromatin is viewed by negative contrast or after metal shadowing. The useful resolution of the secondary electron micrographs, for a suitably mounted specimen, is shown to be as good as that of transmission micrographs of platinum-carbon replicas of the same material.

Animals↗

PubMed1984

The freeze fracture technique in inner ear research.

Freeze-fracture studies on the inner ear have been focused mainly on the normal structure of junctions sealing the endolymphatic compartment, the compartmentalization of the stria vascularis and the junctional stability of the hair cells towards adjacent supporting cells. The hair cells have a very tight type of zonulae occludenetes as compared with other non-sensory epithelia in the inner ear. In contrast to other epithelial cells, the mature hair cells are in most species lacking gap junctions. During embryonic development a loss of gap junctions is an early and significant feature of cells differentiating into future hair cells. The tight junctions in the secretory epithelia (stria vascularis in the cochlea and dark cells around vestibular organs) are morphologically mature before the onset of the ionic maturation of endolymph. Freeze-fracture studies on inner ear pathology are few. The structural alterations of tight junctions in the diseased inner ear are minimal. The functional significance of such small morphological derangements is not known.

Animals↗

PubMed1985

Freeze-fracture studies of human blood platelets activated by thrombin using rapid freezing.

In this study the influence of thrombin activation on human blood platelets has been followed by freeze-fracturing electron microscopy using rapid freezing in order to catch the initial changes in shape and the morphological alterations during the process of exocytosis of secretory granules. We found that isolation of the platelets by itself leads to some degree of shape change, which made it impossible to study the resting discoid platelet by rapid freezing. Activation of the platelets by thrombin induced dilation of the "surface connecting system (SCS)" with formation of large vacuoles as a result of fusion of the secretory granules with SCS. No intermediary fusion stages or structures were observed even using rapid freezing. Volcano-like protrusions and the corresponding complementary pits were seen at the SCS. These structures were interpreted by us as fractures through protoplasmic channels crossing the SCS. These channels originate during the swelling of the SCS as a result of the fusion of secretory granules with the SCS.

Blood Platelets↗

PubMed2004

Influence of chemical and freezing fixation methods in the freeze-fracture of stratum corneum.

A comparison between two fixation techniques for freeze-fracture was established. Stratum corneum (SC) samples from pig epidermis were fixed using high-pressure freezing (HPF) and using plunging in propane freezing; the latter after chemical fixation. Then, frozen samples were freeze-fractured, coated with platinum-carbon, and visualized using a high-resolution low-temperature scanning electron microscope and a transmission electron microscope. Our results indicate that the plane of freeze-fracture was different depending on the fixation and freezing methodology used. In the samples frozen by HPF without chemical fixation, the fracture plane laid mainly between the lipid lamellae. However, when chemical fixation and plunging in propane freezing was used, the fracture plane did not show preference to a specific way. Plunging in propane freezing of chemically fixed samples, on the other hand, provides a more homogeneous fracture behaviour. Thus, depending on the methodology used, we can favour a visualization of either lipid or protein domains of the SC. These results could be very useful in future ultrastructural studies in order to facilitate the microscopic visualization and interpretation of the complex images such as those of SC and even of other samples in which different domains coexist.

Animals↗

PubMed1980

Intercellular junctions in the adrenal medulla: a comparative freeze-fracture study.

Observations of freeze-fractured specimens revealed that intercellular junctions in adrenal medulla are different in nature and number according to species. Only gap junctions of diverse size exhibiting characteristic loop-like configurations were found in hamster chromaffin cells. In addition to such gap junctions, polymorphic focal tight junctions occasionally combined with particle clusters or small gap junctions were found in guinea-pig. So far, no intercellular junctions were found in rat. Discussion is focused on the possible function of these junctions, in keeping with their presumably high lability.

Adrenal Medulla↗

PubMed1978

Communicating junctions of the human sensory retina. A freeze-fracture study.

Using the freeze-fracture technique communicating junctions could be identified between the cells of the adult human sensory retina. Communicating junctions with particles arranged in linear or circular rows as well as in small aggregates were found between adjacent photoreceptor endings. Communicating junctions with particles forming plaques were localized in the inner plexiform layer. They were isolated between unidentified cell processes or lay close to active sites of chemical synapses, thus suggesting the occurrence of "mixed" (chemical and electrical) synapses in this layer. Furthermore, communicating junctions were detected between portions of Müller's cells.

Cell Communication↗