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Results for “Freeze Fracturing”

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PubMed1975

Freeze-fracture replication of junctionsl complexes in unincubated and incubated chick embryos.

Junctional complexes have been investigated in the epiblast of young chick embryos by examination of freeze-fracture replicas and of sections of comparable specimens stained with lanthanum nitrate. By means of freeze-fracture, tight junctions were shown to be present in the unincubated embryo (stage 1 of Hamburger and Hamilton). The number of ridges or grooves was found to vary between 2 and 10 near the dorsal border, whereas isolated ridges were found more ventrally. Lanthanum was unable to penetrate between the cells in the region of the dorsally situated tight junctions. Similar tight junctions were found in incubated embryos (stage 3) examined by both techniques. Tight junctions were also seen in cleavage (pre-laying) embryos examined in section. Gap junctions were extremely uncommon in unincubated embryos, though occasional aggregates of gap junction particles were seen on the lateral cell membranes close to the dorsal surface. In only one instance were associated pits visible. By contrast, gap junctions were more frequently encountered by stage 3, and these junctions possessed both pits and particles. Desmosomes were never seen in the freeze-fracture replicas at either stages 1 or 3, though structures which might be developing desmosomes were visible in sections. The functions of both the tight and gap junctions in the young chick embryo are discussed. The results are also considered in relation to recent theories about the way in which gap junctions are formed.

Animals↗

PubMedDate not supplied

Cholesterol content of focal opacities and multilamellar bodies in the human lens: filipin cytochemistry and freeze fracture.

Adult human lenses with focal opacities were processed for normal as well as freeze fracture electron microscopy. Cholesterol was demonstrated using filipin cytochemistry. Filipin cytochemistry in combination with freeze fracture revealed that the amount of cholesterol in the normal fibre membranes was fully comparable with the amount of cholesterol in the deviating membranes of the focal opacities and the multilamellar bodies. As regards the cholesterol/phospholipid ratio, the deviating membranes have the same state of maturation as the surrounding normal lens fibre membranes, which implies that their synthesis is as highly coordinated as the synthesis of the normal lens fibre membranes.

Cataract↗

PubMedDate not supplied

Freeze-fracture study of Trichomonas vaginalis.

The freeze-fracture technique was used to analyse the organization of the plasma membrane, as well as membranes of cytoplasmic organelles, of the pathogenic protozoan Trichomonas vaginalis. Rosettes formed by 4 to 14 intramembranous particles were seen on the fracture faces of the membrane lining the anterior flagella as well as in fracture faces of the plasma membrane enclosing the anterior region of the protozoan and in cytoplasmic organelles. Special organization of the membrane particles were also seen in the region of association of the recurrent flagellum to the cell body.

Animals↗

PubMed1977

Freeze-fracture of monolayer cultures.

This paper describes a simple method for the freeze-fracturing of cells in monolayers or multi-layer tissue cultures. The method produces high quality replicas and is applicable to the study of virtually any tissue culture or organ culture system. It uses standard materials and equipment for both tissue culture and freeze-fracturing.

Cell Line↗

PubMed1992

Ultrastructural imaging of freeze-fractured plant cells in the scanning electron microscope.

The application of the freeze-fracture and cytoplasmic maceration technique in ultrastructural studies of plant cells is described. A major advantage of the technique is, that by extracting mobile cytoplasmic components from the freeze-fractured cells, surface relief is introduced and three-dimensional information is obtainable. The details of specimen preparation are described and the results obtained are reviewed. The use of chitosan embedding for very small or fragile specimens is described.

Freeze Fracturing↗

PubMed1985

Analysis of desmosomal intramembrane particle populations and cytoskeletal elements: detergent extraction and freeze-fracture.

We have examined sections and freeze-fracture replicas of Triton X-100 detergent-extracted desmosomes from murine palatal epithelium. After extraction of lipids as well as soluble proteins, a cytoskeletal framework remained which consisted of intermediate filaments, microfilaments, and intact desmosomal skeletons. Traversing filaments, which link the intermediate filaments to large intramembrane particles of the P-face, appeared undisturbed within the desmosomal skeletons. Compared to unextracted controls, extracted specimens displayed P- and E-face desmosomal intramembrane particles which were more fully exposed. A broad range of sizes and shapes was apparent for the P-face associated particles. E-face particles, some of which were exposed for the first time, were more homogeneous and generally smaller. Statistical data gathered from a large sample of P- and E-face particle diameters disclosed significant differences among the populations of the two faces. Both fracture faces of extracted desmosomal domains displayed a residual surface upon which the exposed particles seemed to remain lodged. The newly revealed structural features are presented in an hypothetical molecular model which provides for both vertical and horizontal stabilization of desmosomal subcomponents. The model may ultimately be relatable to emerging biochemical characterization.

Animals↗

PubMed1991

Giardia duodenalis: a freeze-fracture, fracture-flip and cytochemistry study.

The freeze-fracture technique was used to study the structural organization of the membranes of trophozoites of the protozoon Giardia duodenalis. No special array of intramembranous particles was observed in the membrane lining the protozoon body or the flagella. A large globular protuberance located in the ventral region displayed several small circular indentations similar to those seen in the dorsal region. These also occurred on the parasite surface as revealed in fracture-flip replicas. A large number of vesicles were observed below the plasma membrane; they corresponded to an acidic compartment as indicated by fluorescence microscopy of acridine orange-stained cells and contained acid phosphatase as indicated by cytochemistry. In addition, gold-labeled macromolecules (albumin, peroxidase, transferrin, and low-density lipoprotein) accumulated in the vesicles. These observations suggest that the peripheral vesicles of trophozoites are part of the endosomal-lysosomal system of G. duodenalis.

Acid Phosphatase↗

PubMed1994

Effects of soman on cerebral astrocyte plasma membranes: a freeze-fracture study.

Astrocytes are identifiable on freeze-fracture by regular geometric aggregates of uniform intramembranous particles (IMP), the 'orthogonal arrays' (OA). These OAs are considered to be potassium channels that maintain the ionic balance around neurons. After subcutaneous administration of a single near LD50-dose of soman in rats, neurotoxic epileptogenic irreversible cholinesterase inhibitor, replicas showed a significant decrease in OA density in the convulsed animals. This dissociation by soman confirms that OA are a particularly labile membranes specialization. Frequent clumping of IMP and numerous cleavage planes were observed; this could be the result of an interaction between the liposoluble poison and membrane phospholipid fluidity.

Animals↗

PubMed1982

Changes in the organization of the outer membrane of Proteus mirabilis during swarming: freeze-fracture structure and membrane fluidity analysis.

Freeze-fracture studies of short, nonswarming Proteus mirabilis revealed the characteristic gram-negative profile of fractured inner membrane with densely packed particles and sectioned outer membrane with little or no fracture plane. Long swarming cells, however, fractured easily along both the inner membrane and a second membrane, probably the outer membrane. The inner membrane had a typical profile, whereas the outer membrane had fewer but more prominent particles. Isolation and purification of the inner and outer membranes of the short and long bacteria and examination of them with electron paramagnetic resonance measurements after spinlabeling supported the above observations. The outer membrane of swarmer cells allowed higher mobility of the spin label than did the outer membrane of the nonswarming short cells, which showed a typical rigid profile. These results suggest that regions of lipid bilayer appear in the outer membrane during swarmer formation. Previous observation of the behavior and biochemistry of P. mirabilis during swarming are discussed in light of these results.

Cell Membrane↗

PubMed1974

Freeze-fracture of microtubules and bridges in motile axostyles.

A freeze-fracture study of the motile axostyles of the flagellate protozoa Saccinobaculus and Pyrsonympha has been undertaken in order to obtain a view of the relationships of microtubules and their cross bridges not dependent on conventional preparative procedures. Reactivation studies using isolated axostyles prepared for freeze-fracture and then thawed demonstrate that we are observing the structure of a potentially functional axostyle. Cross fractures through the axostyle demonstrate more extensive interrow bridging than expected on the basis of observations of thin-sectioned material. Each microtubule has approximately sixfold bridge-binding sites with connections to as many as four interrow bridges. Measurements of microtubule diameter and spacing are significantly larger than those made from sectioned material and may indicate that conventional processing for electron microscopy results in the loss of structurally important water within the microtubule in addition to loss of intertubule material. Longitudinal fractures through the axostyle at various orientations demonstrate a minimum longitudinal periodicity of 160 A for both the spacing of the globular subunits within the microtubule wall and the spacing of the intrarow bridges. While intrarow bridges are strictly periodic and always oriented in parallel, interrow bridges are not strictly periodic and can be oriented at varying angles to the microtubule axis.

Eukaryota↗

PubMed1983

Fine structure of secretory ameloblasts in kitten tooth germs, with special regard to intercellular junctions as revealed by freeze-fracture.

Using both thin sectioning and freeze-fracture replication, junctional complexes at both proximal and distal ends of the cells consisted of tight junctions in close association with gap junctions and desmosomes. The tight junctions generally consisted of smooth, continuous rows of particles on the P-face and corresponding patterns of shallow grooves on the E-face of cell membranes. Though sealing of paracellular spaces around the ameloblasts in the proximal junctional complex was incomplete, there was complete sealing around the ameloblasts and well-developed meshwork structures of tight junctions in distal junctional complexes. Discontinuous and free-ending strands of tight junctions were frequent in junctional complexes, suggesting that ameloblast distal junctional complexes serve, not only as a barrier to high molecular passive substances through the ameloblast layer, but also as a channel for ions and low-molecular substances. Ameloblasts were firmly connected with stratum intermedium cells by desmosomes and gap junctions. The gap junctions on ameloblast basal and lateral surfaces probably function in intercellular transfer of ions and low-molecular substances between the stratum intermedium and ameloblasts and in control of ameloblast cytodifferentiation.

Ameloblasts↗

PubMed1984

Intercellular junctions in the cells of the human enamel organ as revealed by freeze-fracture.

Examined by thin sections and freeze-fracture replication techniques, secretory ameloblasts possessed two sets of the junctional complexes at both proximal and distal ends of the cell bodies, which consisted of tight junctions and occasional gap junctions and desmosomes. The proximal tight junction was fascia occludens, whereas the distal tight junction was zonula occludens. Between adjacent ameloblasts, mature gap junctions were frequent. The stratum-intermedium cells were connected to each other and to the stellate-reticulum cells and ameloblasts by well-developed desmosomes, gap junctions and fascia or macula-type tight junctions. Stellate-reticulum cells were inter-connected by many extensive cytoplasmic processes, in which well-developed desmosomes, small gap junctions and occasional macula-type tight junctions appeared. Thus fascia or macula-type tight junctions as well as many desmosomes seem to serve in mechanical, cell-to-cell adhesion during tooth formation. Frequent and large gap junctions between adjacent stratum-intermedium cells and between the stratum intermedium and the base of the ameloblast suggest that, in relation to enamel formation, these two cell layers form a functional unit.

Ameloblasts↗

PubMed1983

Fine structures of sponge cell membranes: comparative study with freeze-fracture and conventional thin section methods.

Freeze-fracture replicas of sponge cell membranes revealed in general a low density of intramembranous particles, with the exceptions of the membrane (silicalemma) surrounding the siliceous spicules in Ephydatia and the membranes of spherulous cells in Chondrosia. In addition, several types of particle arrangements were observed. A classical necklace is present at the base of the choanocyte flagellum. Rosettes of particles are particularly obvious in the apical membranes of choanocytes, where they are associated with the fuzzy coat covering these cells. Parallel ridges of particles were observed along the microvilli of the choanocyte collar, at sites of insertion of connecting filaments. Rows of particles were observed in the plasma membrane of pinacocytes in Ephydatia where they are located on areas deformed by protruding fibrillar inclusions. Pinacocyte plasma membranes in this species also can contain accumulations of particles which are likely related to desmosomes. Single rows of aligned particles and double rows of staggered particles (sometimes organized in large plates) in addition to rhombic particle arrays were encountered on replicas of marine sponge cell membranes. No classical arrangements corresponding to gap junctions, tight junctions or septate desmosomes were observed. The significance of these data is analysed.

Animals↗

PubMed1987

Monomer-oligomer equilibrium of bacteriorhodopsin in reconstituted proteoliposomes. A freeze-fracture electron microscope study.

An improved freeze-fracture electron microscope procedure has been developed and applied to the study of the association of bacteriorhodopsin in large proteoliposomes reconstituted by reverse-phase evaporation with egg lecithin. Due to the improved accuracy and resolution of this procedure, intramembrane particles, the diameter of which (4.5 nm) closely matched that of bacteriorhodopsin monomer, could be observed at high lipid to protein ratios (greater than or equal to 40 w/w). At lower lipid to protein ratios, larger particles (diameter 7.5 nm) progressively appeared, resulting in bimodal particle size distributions up to a lipid to protein ratio of 1, where the large particles were the sole species present. These large particles were interpreted as corresponding to bacteriorhodopsin oligomers. Because of the large size and homogeneity of proteoliposomes, accurate particle density measurements could be performed. These confirmed the occurrence of a lipid to protein ratio-dependent bacteriorhodopsin monomer-oligomer equilibrium and further allowed us to identify the oligomer as a trimer or a tetramer. In complementary experiments, it was found that the bacteriorhodopsin monomer and oligomer had identical visible CD spectra and light-induced proton pumping rates. However, a large increase of the proton passive leak rate of proteoliposomes was found to be associated with oligomer formation. The appearance of these oligomers may be important as the first step in the formation of two-dimensional crystals of bacteriorhodopsin.

Bacteriorhodopsins↗

PubMed1980

Qualitative and quantitative freeze-fracture studies on olfactory and nasal respiratory structures of frog, ox, rat, and dog. I. A general survey.

A comparative study using freeze-fracturing has been made of surface structures of olfactory and nasal respiratory epithelia of frog, ox, rat and dog. Special attention has been paid to cilia and microvilli present at these surfaces, although the observations include various other structures such as small intracellular vacuoles present in the olfactory receptor endings and infrequent brush cells. Within the mucus overlying the olfactory epithelium membranous vesicles, often attached to olfactory cilia, are seen. Some of these show intramembranous particle distributions similar to those of the rest of the cilia, whereas others are devoid of particles. Smooth vesicles are also found in the mucus of other types of epithelium (respiratory epithelium and Bowman's glands). The freeze-fracture morphology of intracellular secretory vacuoles present in olfactory supporting, Bowman's and respiratory glandular cells of the frog is similar in all these epithelia. Quantitative comparisons are made of the different structures of interest. When corrected for cilia which were not observed, mammalian receptor endings bear 17 cilia on average, whereas frog receptor endings have 6 cilia. The relative magnitudes of the diameters of the cilia and microvilli are, except for frog, the same for all species studied. Dimensions of other structures, e.g., axons, dendrites and dendritic endings are compared in the various species. Freeze-fracture diameters are usually larger than those seen by techniques using dehydration. Dendritic ending densities range from 4.5 X 10(6) (frog) to 8.3 X 10(6) (dog) endings per cm2. Possible sex-dependent differences are only found for these densities and dendritic ending diameters.

Animals↗

PubMed1974

Membrane differentiations in freeze-fractured mammalian sperm.

A correlated thin-sectioning and freeze-fracturing study has been made of guinea pig and rat spermatozoa. In sections, the cell membrane over the acrosome has a concanavalin A and ruthenium red reactive glycocalyx which exhibits an ordered pattern related to the lattice of crystalline domains within the plane of the membrane revealed by freeze-fracturing. The cleaved acrosomal membrane also shows a finer linear periodicity in some areas. The membrane over the equatorial segment of the guinea pig acrosome is marked by a palisade of oblique ridges not observed in the rat. The plasmalemma of the postacrosomal region is rich in membrane intercalated particles, many randomly dispersed, others clustered in rectilinear arrays. A particle-poor zone is found just anterior to the posterior ring. The fold of redundant nuclear envelope posterior to the ring has many nuclear pores in close hexagonal array. The nuclear envelope lining the implantation fossa is devoid of pores. When cleaved it has a particle-free central area surrounded by a broad zone of large, closely packed, hollow particles. The membrane of the mid-piece in the guinea pig (but not the rat) contains linear strands of 6-8-nm particles oriented circumferentially. The membrane investing the principal piece exhibits the usual randomly distributed particles but in addition, a double row of larger (9 nm) particles runs longitudinally within the membrane over outer dense fiber 1. In the corresponding position in thin sections a local thickening of the membrane is discernible. These observations form a basis for further studies on the functional correlates of these regional specializations of the sperm membrane.

Animals↗

PubMed1982

Freeze-fracturing of oral epithelial cells in pemphigus.

Biopsies from the oral mucosa of three patients (two with pemphigus vulgaris and one with pemphigus erythematosus) were taken for both freeze-fracturing and thin-sectioning studies. Oral mucosa from a clinically healthy volunteer was used as control. The samples collected from both the involved and uninvolved oral mucosa of pemphigus patients showed widened intercellular spaces and fewer than normal desmosomes. Thin sections showed a random distribution of tonofilaments. However, in freeze-fracture replicas tonofilaments were arranged in thick bundles with a slightly coiled orientation. In freeze-fracture replicas of samples from a pemphigus patient in remission no difference was found in the number of particles on both fracture faces.

Adult↗

PubMed1978

Freeze-fracturing studies of mitochondrial myopathy. A correlated clinical, biochemical and morphological investigation.

Freeze-fracture studies of pathologically changed mitochondria in situ from muscle biopsies of a 9.5-year-old girl with a mitochondrial myopathy were correlated with clinical, biochemical and histochemical investigations. In the ultrathin sections giant mitochondria with densely packed cristae membranes - often reoriented to concentric circles - and, in addition, paracrystalline mitochondrial inclusions were found. The freeze fracture faces of such transformed mitochondria and preparations of their inner and outer membranes provided a morphological insight in the macromolecular structure of the mitochondrial membrane under such pathological conditions. The results lead to the hypothesis that part of the transformed mitochondria stay active functionally for an extended period by maintaining the delimitation from the cytoplasm and by preserving the macromolecular membrane architecture. This hypothesis could explain the slow progression of the myogenic symptoms.

Child↗