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Freeze-fracture studies on the synapse between the type I hair cell and the calyceal terminal in the guinea-pig vestibular system.

The apposition between type I hair cells and the calyceal terminals of vestibular ganglion cell peripheral processes was studied in the vestibular epithelium of the guinea-pig, using thin-sectioned and freeze-fractured specimens. Chemical synaptic junctions were exceedingly rare in thin-sectioned specimens, and were not seen in freeze-fracture replicas. Furthermore, no gap junctions were present between the hair cell and the calyx. There were, however, regions along the apposition where the membranes were closely apposed. At these regions, the hair cell was invaginated by cytoplasmic protrusions of the calyx and the plasmalemmata of the two cells were separated by only 6-7 nm. The number and conformation of the close appositions varied between different cells. In freeze-fracture replicas, the closely-apposed plasmalemmata of the hair cell and the calyx had no special distribution of intramembrane particles on either membrane leaflet. However, on the external membrane leaflet of the hair cell, a large patch of widely-spaced, large particles surrounded the regions of close apposition. The corresponding region of the plasmalemma of the calyx had no special distribution of particles on either membrane leaflet. The scarcity of chemical synaptic junctions, the absence of gap junctions between the cells and the unique arrangement of particles in the hair cell plasmalemma surrounding regions of close membrane apposition may indicate an unusual mode of synaptic transmission between the type I hair cell and the calyx.

Animals↗

Negative staining of freeze-fractured envelopes of Escherichia coli K12.

Envelope fragments of E. coli K12 have been produced by freeze-fracturing "by hand" and negatively stained after thawing. The outer leaflet of the plasma membrane disintegrated upon thawing whereas the outer leaflet of the outer membrane did not. Negative staining revealed the following structural features on the outer membrane fragments: (i) "grooves" 4-6 nm wide, (ii) spherical particles 6-8 nm in diameter, (iii) "black dots" 3-8 nm in diameter. Treatment of cells with EDTA before freeze-fracturing caused dilation of grooves into holes eventually leading to fragmentation of the outer membrane. A mutant strain deficient in two outer membrane proteins fractured exclusively through the outer membrane. The outer leaflets so obtained disintegrated upon thawing similarly as observed for the outer leaflet of the plasma membrane.

Cell Membrane↗

Assessment of ultrastructure in isolated cochlear hair cells using a procedure for rapid freezing before freeze-fracture and deep-etching.

Separated cochlear outer hair cells and isolated strips of organ of Corti containing hair cells and supporting cells have been rapidly frozen before freeze-fracture and deep-etching by immersion of samples sandwiched between two copper plates into liquid nitrogen-cooled propane: isopentane. Assessment of this procedure has shown that no significant freezing damage occurs. The ultrastructure of the hair cells revealed by freeze-fracture of these non-chemically fixed preparations was generally very similar to that seen in fixed material. This indicates that the processing of cochlear tissue normally used for electron microscopy produces few obvious structural artefacts. It also demonstrated that procedures for isolating cochlear hair cells generally do not affect cell structure significantly. However, some isolated hair cells did show abnormalities within the membranes of the lateral cisternae. Such membrane alterations, which would not be identified by light microscopy, occurred to a variable extent but were more commonly present after prolonged periods in maintenance medium. Deep-etching of the preparations to examine extracellular features around stereocilia revealed clearly lateral cross-links between stereocilia. However, tip-links could not be positively identified in either unfixed or prefixed preparations.

Animals↗

Calorimetric and freeze fracture analysis of lipid phase transitions and lateral translational motion of intramembrane particles in mitochondrial membranes.

Differential scanning calorimetry combined with freeze fracture electron microscopy reveals that thermotropic lipid phase transitions and lateral translational motion of intramembrane particles occur in both membranes of whole, intact rat liver mitochondria and in isolated inner and outer membranes. The onset temperature of the liquid crystalline to gel state lipid phase transition in whole mitochondria and in the isolated outer membrane fraction is biphasic with an initial transition exotherm occurring at 9 degrees C. The onset temperature of the transition exotherm of the isolated inner membrane occurs at -4 degrees C. The onset temperature of the lipid transition endotherm is -15 degrees C for whole mitochondria, the inner membrane, ane the outer membrane fractions. These calorimetric analyses reveal that the bilayer lipid in the inner, energy transducing membrane is more fluid than in the outer membrane. Mitochondrial membranes cooled to temperatures in the region of their transition exotherms and then frozen reveal striking lateral separations between smooth, intramembrane particle-free regions (rich in gel state lipid) and particle-dense regions (rich in integral proteins) in their hydrophobic fracture faces. Such thermotropic lipid-protein lateral separations are completely reversible. These freeze fracture observations suggest that both mitochondrial membranes are naturally fluid to the extent that the integrat membrane proteins can diffuse laterally in the bilayer lipid.

Animals↗

A freeze-fracture electron microscope study of Trichomonas vaginalis Donné and Tritrichomonas foetus (Riedmüller).

Two strains of Trichomonas vaginalis, JH162A , with low pathogenicity, and Balt 44, with high pathogenicity, as well as one highly pathogenic strain, KV-1, of Tritrichomonas foetus were studied by freeze-fracture electron microscopy. The protoplasmic faces ( PFs ) of the cell membranes of all three strains of both species had similar numbers of intramembranous particles (IMPs); however, the particles in the external faces (EFs) of these membranes were least abundant in Trichomonas vaginalis strain Balt 44 and most numerous in those of strain JH162A of this species. In Tritrichomonas foetus strain KV-1 the number of IMPs in the EF was close to but somewhat lower than that in the mild strain of the human urogenital trichomonad . In both species, the anterior, but not the recurrent, flagella had rosette-like formations, consisting of approximately 9 to 12 IMPs on both the PFs and EFs. The numbers and distribution of the rosettes appeared to vary among different flagella and in different areas of individual flagella of a single organism belonging to either species. The freeze-fracture electron micrographs provided a more complete understanding of the fine structure of undulating membranes of Trichomonadinae , as represented by Trichomonas vaginalis, and of Tritrichomonadinae (the Tritrichomonas augusta -type), as exemplified by Tritrichomonas foetus, than was gained from previous transmission and scanning electron microscope studies. Typically three longitudinal rows of IMPs on the PF of the recurrent flagellum of Trichomonas vaginalis were noted in the area of attachment of this flagellum to the undulating membrane. The functional aspects of the various structures and differences between certain organelles revealed in the two trichomonad species by the freeze-fracture method are discussed.

Animals↗

Measurements on the internal structure of freeze-fractured cardiac plasma membrane.

We describe a quantitative analysis of the internal structure of cardiac plasma membranes freeze fractured in situ, including the P-face particle density (lambda), the P-face particle diameter (d), the percent of fracture face area occupied by particles (Ap), and the spatial distribution of particles (random, clustered, or ordered). This analysis has been applied to seven sheep hearts to compare the plasmalemmal internal structures in ventricular and atrial myocytes and Purkinje strands of the same hearts and also to myocytes of frog, chicken, rabbit, and rat ventricles (to compare internal plasmalemmal structure of different vertebrate classes). Measurements were made on tissues conventionally prepared for freeze fracture by glutaraldehyde fixation and cryoprotection. We found that, in the same sheep hearts, lambda and Ap for ventricular plasmalemma significantly exceeded those for atrial plasmalemma and that the distribution of atrial P-face particles was more clustered than that for ventricle. d and Ap for frog ventricular plasmalemmal P-face significantly exceeded values for some of the other vertebrates.

Animals↗

Maturation changes of the plasma membrane of rat spermatozoa observed by surface replica, rapid-freeze and deep-etch, and freeze-fracture methods.

Rat spermatozoa from the epididymis and ductus deferens were observed by surface replica, rapid-freeze and deep-etch, and conventional freeze-fracture methods. By the surface replica method, parallel periodical ridges were observed in the acrosomal region of the spermatozoa from the distal part of the cauda epididymis (zone 6) and from the ductus deferens. The periodicity of the ridges forming a domain was about 35 nm. A quantitative analysis of the spermatozoa along the reproductive tract indicated that 39.4% and 73.5% of the population in zone 6 of the epididymis and in the ductus deferens, respectively, had the domain. None of the spermatozoa from zone 1 through zone 5 had the domain. The results of the rapid-freeze and deep-etch procedure showed that the ridges observed by the surface replica method consisted of linear arrangements of elliptical particles on the ES face of the plasma membrane. The particles were about 30 nm in length and 15 nm in width. On the corresponding PF face of the plasma membrane, linear arrangements of the intramembrane particles (IMPs) of about 8 nm in diameter were observed by both the deep-etch and freeze-fracture methods. The IMPs tended to run in paired parallel lines. A close relationship was observed between the lines of the elliptical particles on the ES and of the IMPs on the PF faces. The elliptical particle may be a protruded part of the IMP(s) or other protein(s) bound to the IMP(s).

Animals↗

Freeze fracture morphology of the tight junctions of the eccrine sweat gland from patients with cystic fibrosis.

The resorption of Na+ and Cl- across the duct of the human eccrine sweat gland is markedly decreased in individuals with cystic fibrosis (CF). Conceivably, a defective transcellular ion transport mechanism or an increased paracellular backflux of ions could account for the abnormal salt resorption in the sweat gland duct and other organs affected in CF. Tight junctions are thought to regulate paracellular ion flow. Specifically, the number of junctional elements observed by freeze fracture are believed to correspond with the extent of paracellular transport. We compared the freeze fracture morphology of tight junctions of eccrine sweat glands taken from 11 control and seven CF patients. In an attempt to "fingerprint" the junctions morphometrically, the following parameters were measured: the number of strands, the depth of the junction from the apical to the basal strand, the angle of intersection between strands, and the mean distance along a strand between intersections with two other strands. No significant difference was observed between control and CF sweat glands in the freeze fracture morphology of the tight junctions of the duct, the segment where the net reabsorption of Na+ and Cl- is abnormally decreased in CF. Significant changes were observed, however, in the means of the number of strands, the depth, and the distance between intersections for the tight junctions of the intercellular canaliculus of the secretory coil, which appears to function normally in CF.

Adolescent↗

Electron microscopic studies of the morphology of the corneal stroma in the rabbit cornea using the freeze-fracture technique.

The morphology of the corneal stroma in the rabbit cornea was investigated under the electron microscope using the freeze-fracture technique. The collagen fibrils were closely packed into collagen bundles. In the freeze-fracture replicas the amorphous ground substance could not be demonstrated. At the stroma membranes were shown with crater-shaped depressions on the P-face and corresponding elevations on the E-face. Regarding the topographical relationships between the membranes and other parts of the fibroblasts, it was concluded in this investigation that the crater-shaped structures probably constitute pores in the plasma membrane of fibroblasts.

Animals↗

Intercellular junctions of the hen parathyroid gland. A freeze-fracture study.

The fine structure of the intercellular junctions of the hen parathyroid gland was studied using freeze-fracture replicas and thin sections. In the conventional thin sections, desmosomes, intermediate junctions (maculae adherentes) and gap junctions were observed, and in the lanthanum-fixed sections, tight junctions (maculae occludentes) were demonstrated as well. In the freeze-fracture replicas, desmosomes, gap junctions, tight junctions and combination forms of gap and tight junctions occurred, but intermediate junctions were not identified. Junctional complexes (zonulae occludentes) were not encountered in any preparations. The gap junctions varied in size and shape; they ranged from irregularly shaped, minute assemblages of particles to large aggregations of a round or elliptic outline. Both the tight junctions and the combination forms of gap and tight junctions also exhibited a variety of shape and dimension, and, depending on the form of the tight junctional strands, they were classified into three types: type I consisted of a simple line of strands; type II consisted of a closed network of strands; and type III consisted of an open network of strands. The combination forms were more numerous than the typical tight junctions. The possible significance of these junctions is discussed in relation to the function of the parathyroid parenchymal cell.

Animals↗

Sampling and transportation of food materials for freeze-fracture from the industrial environment.

A simple holder is described which enables the multiple sampling of food materials for freeze-fracturing in an industrial environment. The holder allows frozen samples to be transferred safely to the laboratory under liquid nitrogen for freeze-fracturing and examination by transmission electron microscopy. The technique has been successfully applied to sampling food from pilot plant and production lines under factory conditions.

Food Handling↗

Ultrastructural organization of freeze-fractured interphase nuclei.

The morphology of intact or membrane-deprived interphase nuclei has been analysed by freeze-fracture electron microscopy. This method appears particularly useful for providing information on the distribution and organisation of chromatin and ribonucleoproteins in the absence of dehydration and embedding artifacts of conventional electron microscope techniques which, among other effects, appear to affect heterochromatin distribution, inducing its aggregation along the nuclear envelope. The main levels of chromatin superstructure, from nucleosome to solenoid fibres, are detectable in the replicas of freeze-fractured nuclei on the basis of the size of their shadow, a parameter particularly suitable for automated image analyses.

Animals↗

Freeze-etching and freeze-fracture structural features of cell envelopes in mycobacteria and leprosy derived corynebacteria.

The structural properties of the cell wall and cell membrane of several mycobacteria and of Leprosy Derived Corynebacteria are investigated by freeze-etching and freeze-fracture. In all cases the freeze-fracture split the cell wall in two asymmetric halves. The cell wall fracture faces of the mycobacteria are characterized by a filamentous network which vary with respect to the amount and complexity among microorganism of the same species and even more of different species. In LDC the structure organization of the cell wall and cell membrane differs from that of mycobacteria. The most stricking difference is the presence on the fracture faces of the LDC cell wall of different classes of particulated entities of yet unknown nature. In the mycobacteria and LDC the periseptal annuli likely provide a potential frame for cell envelope and cell membrane assembly.

Animals↗

Membrane assembly in retinal photoreceptors. II. Immunocytochemical analysis of freeze-fractured rod photoreceptor membranes using anti-opsin antibodies.

We have used a cytochemical technique for labeling freeze-fractured tissues (Pinto da Silva, P., C. Parkison, and N. Dwyer (1981) Proc. Natl. Acad. Sci. U.S.A. 78: 343-347) to examine the distribution of immunoreactive opsin in rod photoreceptor membranes. Aldehyde-fixed retinas of African clawed frogs (Xenopus laevis) embedded in a cross-linked protein matrix were frozen and fractured at -196 degrees C, then thawed and labeled with biotinylated sheep anti-cow opsin IgG followed by avidin-ferritin. In thin sections of plastic-embedded retinas, rod outer segment (ROS) disc membranes exposed by fracturing bound specific antibody intensely and relatively uniformly. However, they differed from membranes of the inner segment as well as those of erythrocytes in that protoplasmic face leaflets did not assume an interrupted bilayer appearance and disc exoplasmic face leaflets were apparently lost during thawing. The disposition of opsin immunoreactivity in the cell membrane was highly asymmetric. Although ROS plasma membranes from which discs are elaborated labeled heavily with anti-opsin after cleavage, fractures passing along inner segment plasma membranes bound very little antibody. In cross-fractures exposing inner segment cytoplasm, we found specific labeling of Golgi complex elements, as well as both perimitochondrial and periciliary vesicles. The latter are presumed to be the vehicle shuttling newly synthesized membrane to the ROS for disc assembly. These results suggest that opsin-containing membrane is sorted out within the cell, being transported from synthetic sites to the immediate periciliary zone where localized insertion into the cell membrane takes place. Furthermore, the close correspondence of the present immunocytochemical analysis with the distribution of opsin deduced from prior quantitative freeze-fracture analysis (Besharse, J. C., and K. H. Pfenninger (1980) J. Cell Biol. 87: 451-463) offers the possibility that fracture-label may be generally useful for study of patterned membrane topography in neuronal cells.

Animals↗

Fusion of mitochondrial inner membranes by electric fields produces inside-out vesicles. Visualization by freeze-fracture electron microscopy.

The fusion of vesicular-shaped mitochondrial inner membranes was observed by a new approach which combines freeze-fracture electron microscopy and electric field-induced fusion. Results show that membrane events caused by the exposure to the electric field can be time-coordinated with sample freezing for subsequent analysis by freeze-fracture electron microscopy.

Animals↗

Freeze-fracture studies of the distal plasma membrane of rat odontoblasts during their differentiation and polarisation.

We have examined freeze-fracture replicas of maxillary first molar tooth germs of newborn rats at early stages of dentinogenesis to study the development of tight junctions in the distal plasma membrane of differentiating odontoblasts. In addition, freeze-fracture was combined with filipin to observe the distribution of cholesterol on the distal plasma membrane of odontoblasts during differentiation. Only gap junctions were present in early differentiating odontoblasts. The distal plasma membrane exhibited low cholesterol content, which might indicate high fluidity. With the beginning of mineral deposition in matrix-vesicles, the first signs of tight junction formation were observed. Further development revealed increasingly complex focal tight junctions. In later stages, when mineralisation is observed progressing to the fibrillar and non-fibrillar constituents of the matrix, well developed focal tight junctions were detected. Concomitantly, cholesterol in distal portions of the odontoblast plasma membrane increased, indicating, probably, a higher rigidity. Thus, a distal plasma membrane domain is established, odontoblasts become fully differentiated, and partial compartmentalisation of matrix occurs. At this stage, odontoblasts may be able to secrete specific matrix molecules to ensure the progression of mineralisation.

Animals↗

Substructure of intercellular junctions in freeze-fractured alveolar-capillary membranes of mouse lung.

The purpose of this study was to examine by freeze-fracture the ultrastructure of intercellular junctions between mouse pulmonary endothelial and epithelial cells, and to relate this fine structure to that deduced from previous physiological and ultrastructural studies using tracer techniques. Junctions between capillary endothelial cells consist of one to three interconnected rows of particles which show occasional discontinuities. Small gap junctions are associated with these rows of particles in the arteriolar end of the capillary bed. At the venular end, the junctions consist of low profile ridges on the protoplasmic fracture (PF) face or complimentary grooves on the exoplasmic fracture (EF) face some of which have a sparse number of associated particles. The vascular junctions are similar to those of vessels in rat omentum and mesentery, and resemble "leaky" junctions described in renal proximal convoluted tubular cells. Tight junctions (zonulae occludentes) between type I pneumocytes or between types I and II pneumocytes consist of a band of interconnecting ridges on the PF face and complimentary interconnecting grooves on the EF face. These continuous epithelial junctions have a structure that is typical of tight occluding junctions. Occasionally zonulae occludentes between type I and type II pneumocytes are discontinuous; this may be the result of cell translocation as pneumocytes are shed into the alveolus. Intravascular perfusion fixation at high pressure (140 cm H2O) had no discernible effect on the structure of endothelial or epithelial junctions. The appearance in our study of freeze-fractured pulmonary endothelial and epithelial junctions reveals clearly the physical basis for the results of ultrastructural tracer and physiological studies which have suggested that it is the alveolar epithelium rather than the endothelium that is the chief permeability barrier to small, water-soluble molecules.

Animals↗

The blood-testis barrier in the toad (Bufo arenarum Hensel): a freeze-fracture and lanthanum tracer study.

Intercellular junctions between Sertoli cells in the toad testis were studied by freeze-fracture and electron-opaque intercellular markers. These junctional specializations are characterized in thin sections by a series of focal fusions on the outer leaflets of both adjacent cell plasmalemmas, associated with bundles of fine filaments in the subjacent Sertoli cell cytoplasms. However, the wide subsurface cisterna of the endoplasmic reticulum, a component constantly associated with Sertoli cell junctions in mammals, is absent in the toad. The intravascularly injected lanthanum hydroxide, used as a tracer compound, gains access to the seminiferous tubules and surrounds spermatogonia and leptotene spermatocytes, but is persistently excluded from germ cells in later stages of development. This indicates that, as is the case in the mammalian testis, a permeability barrier to lanthanum is established which isolates all germ cells beyond leptotene spermatocytes. Freeze-fracture reveals the characteristic occluding junctions between Sertoli cells, but a variation in their geometric patterns was clearly observed in different regions of the toad seminiferous epithelium. The membrane-fractured faces of Sertoli cells embracing differentiating spermatids exhibit a deep junctional complex: up to 50 rows of particles between adjacent Sertoli cells separate these late germ cells from the periphery of the seminiferous tubules. Sertoli cells surrounding early germ cells generally exhibit, instead, a discontinuous, poorly developed network of interconnected rows of particles with few widely spaced strands. This seems to permit the percolation of the intercellular marker in areas of the seminiferous epithelium containing spermatogonia and leptotene spermatocytes.

Animals↗