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PubMed1982

Tissue connections in a transplantable virus-producing sebaceous adenoma of the mouse. II. A freeze-fracture study in conjunction with filipin.

The distribution, size, and configuration of intercellular junctions in the sebaceous tumor of mice were examined using the freeze-fracture technique. Three types of junctions were observed: desmosomes, gap, and tight junctions. Tight junctions in general consisted of short linear unbranched fibrils, and macular or complex tight junctional patterns were present only occasionally. Gap junctions ranged from small sports of 0.9 x 10(-3) micrometer2 to areas of approximately 0.46 micrometers2. Desmosomes were the most frequent junctional specializations, and it is concluded that they are at least partially responsible for this tumor not being metastatic. Filipin in conjunction with freeze-fracture showed filipin-sterol complexes on the plasma membrane, the nuclear envelope, and the membranes of the endoplasmic reticulum and mitochondria. The intercellular junctions were devoid of these complexes indicating that these regions are low in cholesterol.

Adenoma↗
PubMed1986

Morphometric analysis and topological organization of nuclear matrix in freeze-fractured electron microscopy.

The ultrastructural organization of nuclear matrix, purified from intact or membrane-denuded rat liver nuclei, has been analysed by means of freeze-fracturing technique. This method avoids dehydration and embedding which, in conventional thin sectioning, partly distort or mask the matrix ultrastructure. The various matrix components, and mainly the peripheral lamina and the inner network revealed complex arrangements undetectable with conventional techniques. Morphometric analyses performed with a Texture Analysis System (TAS) Leitz, allowed to obtain precise information on the matrix constituents, based on the histograms of their size distribution. These textural characteristics have been utilized in order to identify, by means of a particular computer programme, the putative matrix localization within intact freeze-fractured nuclei.

Animals↗
PubMed1985

Morphology and phase behavior of two types of unilamellar vesicles prepared from synthetic phosphatidylcholines studied by freeze-fracture electron microscopy and calorimetry.

Differential scanning calorimetry and freeze-fracture electron microscopy have been used to characterize the phase behavior and morphology of two types of unilamellar vesicles composed of synthetic phosphatidylcholines. The first type displayed an average diameter of roughly 100 nm and was formed by slow dilution and dialysis of octylglucoside-solubilized lipid. These large, unilamellar vesicles were termed dialyzed, octylglucoside vesicles and could be obtained as a fairly well defined and uniform population of vesicles. The second vesicle type was prepared by a unique procedure involving dialysis of deoxycholate-solubilized lipid at its pre-transition temperature. This procedure produced a much more heterogeneous distribution of vesicle sizes (500 to 4000 nm in diameter) and left some dilamellar and oligolamellar species which could not be conveniently separated from the giant, unilamellar vesicles constituting the major portion of the sample. Both populations of vesicles displayed phase behavior similar, but not identical to that of large, multilamellar vesicles (LMV). Fracture-face morphology of the gel phase was also observed to differ between the two unilamellar and the multilamellar species. LMV have previously been shown to have clear undulated or banded fracture-faces in the P beta phase, while octylglucoside vesicles are shown here to have facetted fracture-faces. Giant, unilamellar vesicles displayed a faint banded morphology similar to but less distinct than that of the LMV P beta phase. These results have demonstrated that bilayer apposition is not required to support the banded fracture-face morphology characteristic of the P beta phase but that a limiting curvature is necessary.

Calorimetry↗
PubMed1976

A simple, selective method for freeze-fracturing spherical cells.

A simple and selective method for freeze-fracturing spherical cells is described. The cells are loaded into the holes of a thin nickel screen. A metal hat is applied to the cell monolayer and the whole assembly, hat-cells-screen, is frozen and then fractured by ripping the hat off. The fractured face on the screen is replicated. By varying the size of the screen holes and by applying the hat to either side of the screen, this method can selectively expose the E face (or the outer half of plasma membrane), the P face (or the inner half of the plasma membrane), or the cytoplasm of the cells. It also provides a means to produce fractures at a preselected area on the cell, if the cells can be loaded onto the screen in an oriented fashion.

Cell Membrane↗
PubMed1988

The ultrastructure of the cysts of Blastocrithidia triatomae Cerisola et al. 1971 (Trypanosomatidae): a freeze-fracture study.

Cysts of Blastocrithidia triatomae from hosts and from in vitro cultures were investigated by freeze-fracture and freeze-etch electron microscopy. No differences in structure were observed between cysts frozen immediately and those fixed and cryoprotected with glycerol. The ultrastructure of all cysts differed considerably from that known for mastigote stages of trypanosomatids: the nuclear pores were surrounded by circular fracture-plane transitions between the inner and outer nuclear membranes; the matrix of the kinetoplast appeared banded; the mitochondrion and the endomembrane system were poorly developed; no flagellar apparatus or basal body could be found; and the subpellicular microtubules characteristic of trypanosomatids were absent. Beneath the surface membrane, a special region, 80-90 nm thick and consisting of several rows of closely packed particles was observed. Intramembrane particle (IMP) distribution on the fracture faces of the surface membrane was highly asymmetric: the ectoplasmic face had very few IMPs, whereas the protoplasmic face contained numerous, closely packed IMPs. No external cyst wall was present.

Animals↗
PubMed1987

Freeze fracture study of the schizonts and merozoites of Leucocytozoon caulleryi.

The fine structure of the schizonts and merozoites of Leucocytozoon caulleryi was studied by the freeze fracture technique. Young schizonts were spherical, surrounded by a thick fibrous capsule and possessed numerous nuclei. The nucleus was surrounded by the outer and inner nuclear membrane. A number of nuclear pores were evident in the nuclear membranes. Advanced schizonts were characterized by the formation of cytomeres. Each cytomere was surrounded by a membrane. Several nuclei lay along the periphery of the cytomere. The protoplasmic face (P-face) of the membrane of the cytomere contained numerous intramembranous particles (IMP). The exoerythrocytic merozoites were surrounded by a two-layered pellicle consisting of the outer and inner membrane. During freeze fracturing, each of the two membranes could split along its hydrophobic interior to yield a total of four fracture faces. The P-faces of the outer and inner membranes were closely covered with evenly distributed IMP. The external faces (E-face) of these membranes had less IMP than the P-faces. The membrane architecture of the erythrocytic merozoites was similar to that of the exoerythrocytic merozoites. The erythrocytic merozoite was enveloped by an additional membrane probably derived from the plasma membrane of host cell.

Animals↗
PubMed1991

Unusual membrane-bound bodies in the basal lamina of the uriniferous tubules of the camel Camelus dromedarius. Freeze-fracture and ultrathin-section study.

Kidney samples of the camel Camelus dromedarius were aldehyde fixed and glycerol impregnated for ultrathin-section and freeze-fracture studies of the basal lamina. Results obtained show the presence of extracellular membrane-bound bodies within the thick basal lamina of the tubular portion of the nephron. The 10- to 500-nm bodies appear isolated and are found at various levels along the width of a highly structural lattice basal lamina. The bodies are observed either in small groups or as single structures which are invariably surrounded by a clear halo of the basal lamina. In ultrathin sections they appear limited by a typical unit-membrane structure, and their interior may appear empty or may exhibit material of variable electron opacity. Freeze-fracture replicas reveal the limiting membrane of the bodies which appear either as concave or convex structures. Intramembrane particles (IMPs) measuring between 5 and 15 nm are present in some of the bodies, whilst others appear devoid of IMPs. The IMPs are present in both concave and convex surfaces and are usually aggregated into clumps. The region of the basal lamina which contains the membrane-bound bodies is usually granular except in the area immediately surrounding the bodies which corresponds to the clear halo observed in thin sections. Although these basal lamina membrane-bound bodies appear to be similar to matrix vesicles previously described in mineralizing tissues, it seems unlikely that they are involved in calcification. It is possible that the membrane-bound bodies and the highly configurated basal lamina may be related to ionic transport mechanisms which are associated with the high osmolarity of the camel urine.

Animals↗
PubMed1977

Ultrastructure of the sodium pump. Comparison of thin sectioning, negative staining, and freeze-fracture of purified, membrane-bound (Na+,K+)-ATPase.

Purified (Na+, K+)-ATPase was studied by electron microscopy after thin sectioning, negative staining, and freeze-fracturing, particular emphasis being paid to the dimensions and frequencies of substructures in the membranes. Ultrathin sections show exclusively flat or cup-shaped membrane fragments which are triple-layered along much of their length and have diameters of 0.1-0.6 mum. Negative staining revealed a distinct substructure of particles with diameters between 30 and 50 A and with a frequency of 12,500 +/- 2,400 (SD) per mum(2). Comparisons with sizes of the protein components suggest that each surface particle contains as its major component one large catalytic chain with mol wt close to 100,000 and that two surface particles unite to form the unit of (Na+,K+)-ATPase which binds one molecule of ATP or ouabain. The further observations that the surface particles protrude from the membrane surface and are observed on both membrane surfaces in different patterns and degrees of clustering suggest that protein units span the membrane and are capable of lateral mobility. Freeze-fracturing shows intramembranous particles with diameters of 90-110 A and distributed on both concave and convex fracture faces with a frequency of 3,410 +/- 370 per mum(2) and 390 +/- 170 per mum(2), respectively. The larger diameters and three to fourfold smaller frequency of the intramembranous particles as compared to the surface particles seen after negative staining may reflect technical differences between methods, but it is more likely that the intramembranous particle is an oliogomer composed of two or even more of the protein units which form the surface particles.

Adenosine Triphosphatases↗
PubMed1998

Freeze-fracture analysis of plasma membranes of CHO cells stably expressing aquaporins 1-5.

Several studies suggest that aquaporin water channels can be identified in membranes by freeze-fracture electron microscopy. For this report, Chinese Hamster ovary cells were stably transfected with cDNAs encoding aquaporins 1-5. Measurement of the osmotic water permeability of the cells confirmed that functional protein was expressed and delivered to the plasma membrane. By freeze-fracture electron microscopy, a 20% increase in intramembrane particle (IMP) density was found in plasma membranes of cells expressing AQP2, 3 and 5, and a 100% increase was measured in AQP1-expressing cells, when compared to mock-transfected cells. On membranes of cells expressing AQP4, large aggregates of IMPs were organized into orthogonal arrays, which occupied 10-20% of the membrane surface. IMP aggregates were never seen in AQP2-transfected cells. Hexagonally packed IMP clusters were detected in approximately 5% of the membranes from AQP3-expressing cells. Particle size-distribution analysis of rotary shadowed IMPs showed a significant shift from 13. 5 (control cells) to 8.5 nm or less in AQP-expressing cells; size distribution analysis of unidirectionally shadowed IMPs also showed a significant change when compared to control. Some IMPs in AQP expressing cells had features consistent with the idea that aquaporins are assembled as tetramers. The results demonstrate that in transfected CHO cells, AQP transfection modifies the general appearance and number of IMPs on the plasma membrane, and show that only AQP4 assembles into well-defined IMP arrays.

Animals↗
PubMed1980

Development of the postsynaptic membrane in Xenopus neuromuscular cultures observed by freeze-fracture and thin-section electron microscopy.

The formation of synapses between cultured neurons and muscle cells from Xenopus embryos has been studied with freeze-fracture and thin-section techniques. Clusters of large P-face intramembranous particles (about 11-12 nm) were observed in both innervated and non-innervated muscle cells. They presumably represented clusters of acetylcholine (ACh) receptors because of their close resemblance to the post-junctional particle clusters at the adult neuromuscular (N-M) junctions. In one-day cocultures, particle aggregates could be observed in more than 50% of the N-M contacts. At this stage, these aggregates were diffusely distributed along the contacts. After two days of coculture, extensive and tight clustering of large particles was seen along the length of persisting N-M contacts. Each particle cluster was composed of many particle aggregates and a particle-free groove demarcated each aggregate from its neighbor, thus producing a convoluted appearance of the membrane, which corresponded well with the thin-section image of the membrane profiles at the N-M contacts. In both freeze-fracture and thin-section images, membrane depressions with a diameter of about 0.1 micron were often observed in the vicinity of N-M contacts in newly innervated muscle cells. Within the pits of these depressions a small aggregate of large particles similar to those in the sarcolemma was often encountered. Such particle-rich membrane depressions were also observed in non-innervated muscle cells. They may represent sites for the incorporation of new ACh receptors in light of current theories. Particle aggregates were also closely associated with certain deep membrane invaginations, suggesting that these structures may be involved in the concentration of ACh receptors. Close membrane contacts were observed between nerve endings and muscle cells in young cocultures thin-sectioned. Gap junction-like particle aggregates were also observed in the muscle membrane along identified young N-M contacts. These data suggest that the formation of transient gap junctions may accompany the initial stages of synaptogenesis in Xenopus N-M cultures.

Acetylcholine↗
PubMed1993

Structural variability of the sub-surface cisternae in intact, isolated outer hair cells shown by fluorescent labelling of intracellular membranes and freeze-fracture.

The intracellular membrane systems in intact, isolated outer hair cells were visualised using the fluorescent membrane probe 3,3'-dihexyloxacarbocyanine iodide (DiOC6) and by freeze-fracture, and f-actin distribution was examined with rhodamine-phalloidin. DiOC6 stained the sub-surface cisternal membranes in the lateral wall and revealed a membrane system running in the centre of the cell from the nucleus to the sub-cuticular region. In optical sections of the lateral wall of fluorescently labelled cells, obtained by scanning laser confocal microscopy, the sub-surface membrane appeared as a fenestrated sheet or a fine network of tubules. Freeze-fracture replicas of rapidly-frozen, unfixed outer hair cells also showed the sub-surface membrane as a fenestrated sheet in some cells or as a network of tubules in others. These combined studies indicate that the interruptions within the cisternal membranes as seen in normal thin sections of outer hair cells are not fixation artefacts but may reflect the dynamic and plastic properties of this membrane system. Double staining of cells with rhodamine-phalloidin and DiOC6 showed substantial co-localisation of intracellular membranes and f-actin. The results suggest there may be a continuous, dynamic endoplasmic reticulum system, forming a core in the centre of the cell, broadening in the subcuticular region and extending down the lateral wall, that may have a role in the turnover and distribution of cytoskeletal assemblies within the outer hair cell.

Actins↗
PubMed1985

Membrane structure of vesiculotubular complexes in developing axons in rat optic nerve: freeze-fracture evidence for sequential membrane assembly.

Intra-axonal vesiculotubular complexes, located within developing axons in the optic nerve of eight-day-old rats, were examined by freeze-fracture electron microscopy. The clusters usually fill most of the cross section of the axon and extend for approximately 1 micron along the fibre axis. As seen in freeze-fracture, the E- and P-faces of the membranes comprising these clusters exhibit a paucity of intramembranous particles (i.m.ps). This i.m.p.-poor membrane structure is different from that of the axolemma per se, which contains i.m.p. densities of ca. 120 micron-2 on the E-face and ca. 400 micron-2 on the P-face. Since earlier studies indicate that the vesiculotubular complexes fuse with the axon membrane so as to contribute to membrane growth, it is suggested that axonal differentiation involves a sequential mode of membrane development, in which an initial growth of a relatively undifferentiated membrane bilayer is followed by in situ insertion of specialized proteins within specific membrane domains.

Animals↗
PubMed1976

Stereological method for estimating relative membrane surface area in freeze-fracture preparations of subcellular fractions.

Microsomal fractions from liver cells are a mixture of vesicles derived from a number of different cellular membranes. These can be differentiated on freeze-fracture preparations by their characteristic density of intramembranous particles. A stereological method is developed which allows the estimation of the relative membrane surface of the various membrane types by using freeze-fracture preparations. The sample is restricted to concave profiles without cast shadow. The numerical frequency distribution of vesicles with respect to particle density is determined. The estimation of relative surface area must consider the effects on sampling of (a) variable size distributions of vesicles and (b) loss of small profiles. Correction coefficients are derived which allow a differential vesicle count to be transformed into an estimate of relative membrane surface.

Animals↗
PubMed1981

Freeze fracture studies of muscle plasma membrane in human muscular dystrophy.

Freeze fracture analysis of intramembranous particle density in skeletal muscle plasma dystrophy from 7 patients with Duchenne muscular dystrophy (DMD), 5 patients with facioscapulohumeral muscular dystrophy (FSH) and 5 patients with myotonic dystrophy (MyD) were carried out. Marked depletion of intramembranous particles including orthogonal arrays was noted in DMD while only orthogonal arrays were significantly decreased in FSH. No abnormalities were noted in MyD.

Adolescent↗
PubMed1978

The cell surface of Trypanosoma cruzi: cytochemistry and freeze-fracture.

The ultrastructure of epimastigotes of Trypanosoma cruzi, obtained from acellular cultures, and bloodstream trypomastigotes, isolated from infected mice, were studied by thin-sectioning and freeze-fracturing techniques. Epimastigotes showed a thin (5 nm) surface coat when stained with ruthenium red, while the surface coat of trypomastigotes was more prominent (15 nm thick). Both P and E faces of the plasma membrane of T. cruzi had roughly the same number of intramembranous particles (IMP) as seen by freeze-fracture. The plasma membrane of bloodstream trypomastigotes had less IMP than epimastigotes. Several differentiations of the plasma membrane was observed. In epimastigotes a cytostome appears as a particle-poor region delimited by a pallisade-like row of adjacent IMP. Bloodstream trypomastigotes did not have a cytostome. Instead, abundant pinocytic vesicles were observed. At the base of the flagellum of epimastigotes a ciliary necklace was found. At this region, the surface coat was differentiated as long, hair-like projections after staining with ruthenium red. The flagellar membrane had less IMP than the body membrane. Clusters of IMP were present on both faces of the flagellar membrane at the flagellar-body adhesion zone of epimastigotes. Linear arrays of IMP were also seen. In bloostream trypomastigotes clusters of particles were observed both on the flagellar and cell body membranes. Our observations demonstrate the presence of considerable structural variations of the T. cruzi plasma membrane at the two stages of the life cycle studied.

Animals↗
PubMed1981

Freeze-fracture studies of muscle caveolae in human muscular dystrophy.

Freeze-fracture studies of caveolar density and distribution were carried out in skeletal muscle plasma membrane from 6 patients with Duchenne muscular dystrophy (DMD), 5 patients with facioscapulohumeral dystrophy (FSH), 5 patients with myotonic dystrophy (MyD) and 5 normal control subjects. The results showed a significant increase in the number of caveolae and a decrease in size in a population of muscle plasma membranes from patients with DMD. No significant changes in the caveolae were observed in muscle plasma membranes from patients with FSH and MyD.

Adult↗
PubMed1981

Freeze-fracture and deep-etching studies on zymogen-granule membranes of the rat pancreas.

Whole pancreatic zymogen granules or their membrane fraction were examined by freeze-fracture or deep-etching under different experimental conditions. The granules were fixed for different time periods, or not fixed, and were cryoprotected with glycerol or DMSO; 3% glutaraldehyde followed by 30% glycerol were finally chosen for giving the best resolution and the highest density of intramembrane particles (IMP). IMP are present on the PF and EF leaflets. Their number decreases with the duration of the fixation. Several granules exhibit IMP-free blebs. Incubation of the granules with protamine sulfate causes an aggregation of IMP and of the rough-textured background on the EF leaflet. A second fracture plane can be formed and has been shown by deep-etching to be intercalated between PF and EF. Deep-etching has also shown that particles attached to the perimeter of the granules and of the blebs are, in fact, large nodules on the PS face which partially extend onto the blebs and do not aggregate with the IMP after protamine treatment. Fusion is also indicated between membrane vesicles. Freeze-fracture of the purified membrane fraction seems to indicate the formation of an IMP cap during the lysis of the granule. Moreover, large nodules remain present on the PS face on these membrane fractions but the majority disappear after washing at pH 11.2 with Na2CO3 and EDTA.

Animals↗
PubMed1986

Morphometric and topologic analysis of freeze-fractured interphase nuclei.

Interphase rat liver nuclei were studied by freeze fracturing followed by electron microscopic observations. This method permits information on the native organization of the nuclear components in the hydrated state to be obtained. Morphometric analyses, performed with a Leitz Texture Analysis System, gave precise information on the different nuclear components, based on the histograms of their size distribution in heterochromatin, interchromatin and nucleolar areas. The textural characteristics were analyzed by computer to determine the topologic distribution of the solenoid chromatin fibers, the nucleosome filaments and the ribonucleoproteins in the different nuclear domains.

Animals↗