Fracture Faces Of Membrane Research Articles

The action potential of Dionaea muscipula Ellis

The intention of this investigation was to acquire more concise information about the nature of the action potential of Dionaea muscipula Ellis and the different types of cells generating and conducting it. It is shown by microelectrode measurements that, besides the sensory cells, all the major tissues of the trap lobes are excitable, firing action potentials with pronounced after-hyperpolarizations. The action potentials are strictly dependent on Ca(2+). Their peak depolarizations are shifted 25-27 mV in a positive direction after a tenfold increase in external Ca(2+) concentration. Perfusions with 1 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or 1 mM LaCl3 completely inhibit excitability. Magnesium ions only slightly affect the peak depolarizations but considerably prolong action potentials. Sodium azide and 2,4-dinitrophenol also abolish excitation, probably by reducing the intracellular ATP concentration. Furthermore, it is tested whether the sensory cells can be distinguished from the other cells of the trap by their electrical behaviour. The resting potentials of sensory cells (-161±7 mV) and mesophyll cells (-155±8 mV) are of the same magnitude. Changes in external ion concentrations affect resting and action potentials in both cell types in a similar way. Additional freeze-fracture studies of both cell types reveal similar numbers and distributions of intramembrane particles on the fracture faces of the plasma membrane, which is most likely the mechanosensor. These findings stress the view that the high mechanosensitivity of the sensory hair results from its anatomy and not from a specialized perception mechanism. It is proposed that trap closure is triggered by a rise in the cytoplasmic concentration of Ca(2+) or a Ca(2+)-activated regulatory complex, which must exceed a threshold concentration. Since the Ca(2+) influx during a single action potential does not suffice to reach this threshold, at least two stimulations of the trap are necessary to elicit movement.

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.

Freeze-fracture study of heart mitochondria in the condensed or orthodox state

Rat heart mitochondria were isolated and forced in a well-defined metabolic state. After freeze-fracturing, the intramembrane particle dimension and density on both fracture faces of the inner mitochondrial membrane were measured. No significant differences could be calculated between the diameter of the membrane particles in the five different states. However, the particle density on the fracture faces of the inner mitochondrial membrane in the condensed configuration is significantly smaller than in the orthodox configuration on the 99.5% level of confidence. These results are compared with the literature, where conflicting data have been published about these particle densities.

Seasonal variations in the fine structure ofHypogymnia physodes (lichenizedAscomycetes) and itsTrebouxia photobiont

The lichenized ascomyceteHypogymnia physodes was collected every second month during a one year period at the same site in a wood near the city of Zurich and investigated with light and electron microscopy techniques. Temperature and relative humidity were measured at the collecting site. Seasonal variations in the germination rate of soredia, in photobiont cell size and cell number, in type and amounts of storage products, and in the density of intramembranous particles of the plasma membrane of theTrebouxia photobiont were observed. Highest germination rates and intense aplanospore formation were observed in January, highest particle densities in both external and internal fracture faces of the plasma membrane in March. Lipids and starch were most abundant in spring to early summer. In summer and autumn numerous photobiont cells died off.

Heliobacterium chlorum: cell organization and structure

The basic cellular organization of Heliobacterium chlorum is described using the freeze-etching technique. Internal cell membranes have not been observed in most cells, leading to the conclusion that the photosynthetic apparatus of these organisms must be localized in the cell membrane of the bacterium. The two fracture faces of the cell membrane are markedly different. The cytoplasmic (PF) face is covered with densely packed particles averaging 8 nm in diameter, while the exoplasmic (EF) face contains far fewer particles, averaging approximately 10 nm in diameter. Although a few differentiated regions were noted within these fracture faces, the overall appearance of the cell membrane was remarkably uniform. The Heliobacterium chlorum cell wall is a strikingly regular structure, composed of repeating subunits arranged in a rectangular pattern at a spacing of 11 nm in either direction. We have isolated cell wall fragments by brief sonication in distilled water, and visualized the cell wall structure by negative staining as well as deep-etching.

Increase in the number of presynaptic large intramembrane particles during synaptic transmission at the Torpedo nerve-electroplaque junction

Small pieces of Torpedo electric organ were treated with 4-aminopyridine. a drug which greatly increases the duration of transmitter release in a single nerve impulse, transforming the normally brief electroplaque potential to a giant discharge. Specimens of tissue were cryofixed by rapid freezing using liquid coolants at precise time intervals during transmission of a single giant discharge, and then examined by freeze fracture. In each experiment, we monitored the electrical response of one specimen during the freezing run to check the physiological responsiveness of the tissue and to determine the precise time of contact with the cryogenic liquid. The general appearance of nerve terminals after cryofixation was similar to that of terminals from chemically fixed and cryoprotected tissue. The major morphological change observed during the time course of the giant discharge was a marked increase in the density of intramembrane particles larger than 10 nm on both the protoplasmic and external faces of the presynaptic membrane. This change appeared in specimens frozen within the first few milliseconds after the stimulus, that is, at a time corresponding to the onset of the rising phase of the potential (3 ms). At the end of the giant discharge, the particle density returned to control values with the same time course as the potential trace. Pits of 20 nm or larger, probably due to vesicle-membrane interaction, were found in a small proportion of nerve terminals. Their occurrence increased only at 120–150 ms after the stimulus, that is a long time after the beginning of the giant potential and of the change in intramembrane particles. The size distribution of particles was also determined in the membrane of synaptic vesicles exposed by cross fracture of terminal boutons; it was found to be similar to that of the unstimulated presynaptic membrane and it did not change during the giant discharge. Stimulation experiments were also earned out in a modified solution containing no added calcium. 20 mM magnesium and 4-aminopyridine. The propagation of impulses along the nerves to the electric organ was not inhibited in the modified solution but acetylcholine release was prevented and no increase in particle density was found on the presynaptic membrane. These and previous biochemical experiments on this tissue suggest that the release of the neuro. transmitter acetylcholine is associated with a transient occurrence of large intramembrane particles on the two fracture faces of the presynaptic membrane. At variance from the results obtained on the frog neuromuscular junction [Heuser et al. (1979) J. Cell Biol. 81, 275] the present changes of intramembrane particles apparently did not follow vesicle fusion. The possible explanations for the discrepancy between the two sets of results have been discussed.

Effect of Hyperthermia on the Plasma Membrane Structure of Chinese Hamster V79 Fibroblasts: A Quantitative Freeze-Fracture Study

Hyperthermia in the range 41-45 degrees C can induce wide biochemical, physiological, and morphological changes in mammalian cells both in vivo and in vitro. In general, its effects on membranes, particularly on the plasma membrane, are still poorly understood. To investigate the effects of heat on this cell structure, Chinese hamster V79 fibroblasts were exposed to 43 degrees C hyperthermia for 1 h, immediately fixed with glutaraldehyde after treatment, and freeze-fractured for electron microscopic examination. Particular attention was given to the density and size of intramembranous particles (IMPs) on both protoplasmic (PF) and external (EF) fracture faces of the plasma membrane. The quantitative study performed by an interactive image analyzer on the IMPs, generally reported as plasma membrane proteins, showed in heat-treated cells a statistically significant increase in their density and size on both fracture faces. The differences observed demonstrate that in our experimental conditions, hyperthermia in plasma membranes produces structural changes whose biological significance has to be clarified. Moreover, our findings seem to support recent data indicating an involvement of membrane proteins in the cell response to hyperthermia.

Freeze‐fracturing at defined temperatures provides information on temperature rise during fracture, and on membrane complementarity

SUMMARYAn apparatus is described which allows freeze‐fracturing at defined temperatures, followed by immediate immersion of the specimen in liquid nitrogen. Replication is carried out in a Bullivant & Ames (1966) freeze‐fracture device.Using yeast as a test specimen, the following results were obtained with the apparatus: (a) It was confirmed that fracturing at temperatures between 243 K and 223 K gave undeformed volcanoes on the PF of the plasma membrane, as shown originally by Steere et al. (1980). (b) Considerable energy is released by the fracturing process, both as shown by thermocouple readings and by the fact that at relatively high fracture temperatures portions of the specimen surface were melted. A temperature rise of 50–70 K was indicated, (c) Under standard conditions, there is a lack of complementarity between the yeast plasma membrane fracture faces, trigonal point particles not being present opposite the corresponding depressions in the array. Fracturing yeast suspended in salt solutions at 203 K demonstrates these particles. Their absence in normal fractures can be explained by ‘secondary fracture’, a concept based on polymer fracture studies.

Detection of carriers of human Duchenne muscular dystrophy by freeze-fracture analysis of erythrocyte plasmalemmal intramembrane particles.

Critical examination of plasma membrane particles on fracture faces of erythrocyte plasma membranes of human obligate carriers of Duchenne muscular dystrophy, control patients, and a patient afflicted with Duchenne muscular dystrophy revealed a 32% decrease in the number of intramembrane particles in erythrocytes of carriers compared with erythrocytes sampled from control patients. These results support the notion that quantitative analysis of intramembrane particles in freeze-fractured erythrocyte plasma membranes represents a new, rapid, simple, and highly accurate diagnostic tool for detection of carriers of human Duchenne muscular dystrophy.

Exocytosis in non-plasmolyzed and plasmolyzed tobacco pollen tubes

Exocytosis occurring during deposition of secondary wall material was studied by freeze-fracturing ultrarapidly frozen non-plasmolyzed and plasmolyzed tobacco pollen tubes. The secondary wall of tobacco pollen tubes shows a random orientation of microfibrils. This was observed directly on fractures through the tube wall and indirectly as imprints of microfibrils on fracture faces of the plasma membrane of non-plasmolyzed tubes. About half of the plasmatic fracture faces from non-plasmolyzed and plasmolyzed pollen tubes carried hexagonal arrays of intramembraneous particles in between randomly distributed particles. Deposition of secondary wall material was often accompanied by an undulated plasma membrane and the presence of membrane-bound vesicles in invaginations of the plasma membrane, between the plasma membrane and secondary wall and-especially in plasmolyzed tubes-within the secondary wall of tube flanks and wall cap. The findings are discussed in connection with published schemes of membrane behaviour during exocytosis.

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

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

Membrane specializations in mammalian lens fiber cells: distribution of square arrays.

Fragments of intact rat, pig and bovine lenses and isolated membranes from bovine lenses were examined by freeze-fracture-etch electron microscopy employing samples ultra-rapidly frozen in the absence of fixatives or cryoprotectants. Complementary replicas of the bovine outer cortex clearly display small patches of square arrays (6.6 nm repeat) as structures distinct from gap junctions in the same membranes. In the outer cortex square arrays appear in single membranes where the extracellular space is not thinned, whereas gap junctions, as in other tissues, only occur where the extracellular space is greatly attenuated. Square arrays in the middle cortex appear as patches of varying size surrounded by smooth membrane fracture faces. Within the undulating membranes of tongue-and-groove interdigitations in the inner cortex and nucleus, the square arrays are extensive and are located specifically on the regions which have convex curvature toward the cytoplasm. Square crystalline regions alternate with non-crystalline regions within each membrane of an undulating pair. Across the extracellular space, crystalline regions are matched with non-crystalline regions, thus making it unlikely that the square array participates in intercellular communication. Because the undulations occur in isolated membranes following urea washing to remove cytoplasmic and extrinsic membrane proteins, the square arrays probably play a crucial role in the formation of the undulations and in the maintenance of membrane curvature. X-ray diffraction experiments show reflections from the crystalline square array for all twenty of our preparations of isolated membranes. These x-ray experiments indicate that the square arrays observed by freeze-fracture are abundant in lens fiber cell membranes.

Rotary-shadowed freeze-fracture replicas: a simple method for the analysis of fracture faces.

In rotary-shadowed freeze-fracture replicas, intramembrane particles on the periphery of a membrane fracture face are not uniformly shadowed from all sides. Those eccentrically positioned intramembrane particles with a net centripetally directed shadowing are on a convex fracture face. In contrast, those eccentrically positioned intramembrane particles with a net centrifugally directed shadowing are on a concave fracture face. Centrally positioned intramembrane particles on convex faces are uniformly shadowed from all sides; however, central depressions of concave faces are often unshadowed.

Ultrastructure of pycnidiospore walls and hyphae of Septoria nodorum

Fine-structural features of pycnidiospore walls and hyphae of Septoria nodorum were studied using thin sections stained with tannic acid, freeze-fracturing and freeze-etching techniques. The wall was composed of two layers, an outer electron-dense layer and an inner electron-transparent layer. Fine-structural features of the walls of hyphae and pycnidiospores appeared to be similar. The fracture faces of the cytoplasmic membrane of pycnidiospores were seen in the freeze-fractured cells. Thin fibrillar and globular material of different sizes partly covered the etched surface of the cell. The fibrillar material was best visualized with the freeze-etching technique. The globular material was clearly revealed using the rotatory shadowing technique.

Complementary freeze-fracture and freeze-etch preparations

Whereas freeze-etching technology was introduced in 1957 (1) and the first freeze-etching equipment to be made available commercially was developed in 1961 (2), the need for complementary replicas was not recognized until Branton (3) proposed that the two membrane faces seen in freeze-etched specimens were not the inner and outer surfaces of membranes but represented two new faces which resulted from the splitting of membranes between the two lipid layers. Several indirect methods were developed to test Branton's hypothesis and most of them provided confirming evidence. It became obvious that complementary replicas could provide the indisputable answer to this theory; wherever one membrane fracture face appeared on one specimen the other fracture face should appear in the same position on the complementary specimen. Several devices were developed to make possible the preparation of complementary replicas (4, 5, 6, 7).

Ultrastructural changes induced by the systemic fungicides triadimefon, nuarimol and imazalil nitrate in sporidia of Ustilago avenae

AbstractThe effects of the systemic fungicides triadimefon, nuarimol, and imazalil nitrate (5 μg ml‐1 for 8 and 16 h) on the ultrastructure of sporidia of Ustilago avenae were studied using transmission and scanning electron microscopes. Thin‐section and freeze‐fracture techniques demonstrated fine structural alterations to the cytology of the fungus. Compared with untreated controls, in which the cells were generally unicellular and uniform in shape and ultrastructure, fungicide‐treated cells exhibited considerable thickening of peripheral cell walls and septa, an increased number of mitochondria that were enlarged and irregular in shape, greater amounts of endoplasmic reticulum, and extensive vacuolisation and accumulation of lipid bodies. The vacuoles were filled with membrane‐like aggregates and vesicles, which could be extruded by exocytosis. The plasmalemma ultrastructure of fungicide‐treated sporidia, observed by scanning electron microscopy after freeze‐fracture was significantly different from that of control cells. The antifungal agents caused hemispherical pits, protrusions, invaginations, and aggregation of intramembrane particles on the membrane fracture faces. Specific structures, on the plasmalemma of plasmatic and extraplasmatic fracture faces, support the assumption of exocytosis. The ultrastructural changes were more pronounced after triadimefon and nuarimol treatment than after imazalil nitrate treatment. An extended incubation time (16 h) with triadimefon and nuarimol resulted in complete destruction of the cellular system of most sporidia.

Freeze-fracture electron microscopy of the plasmalemma of Ustilago avenae treated with systemic fungicides: A quantitative analysis of the intramembrane particles

The interaction of the systemic fungicides triadimefon, nuarimol, and imazalil-nitrate with the plasmalemma of Ustilago avenae was studied using freeze-fracture electron microscopy. These antifungal agents produced alterations in the plasmalemma, such as hemispherical pits and protrusions, deformations of invaginations, and formation of craters or similar structures on the membrane fracture faces. The ultrastructural changes in the plasmalemma were more pronounced after triadimefon and nuarimol treatment than after imazalil-nitrate treatment. Intramembrane particles (IMPs) were aggregated, resulting in particle-free areas which showed some exocytotic activity. A quantitative analysis was performed to determine the topographical distribution of IMPs on the extraplasmatic and plasmatic fracture faces of the plasmalemma. The evaluation revealed statistically significant differences in the membrane structure of untreated and fungicide-treated sporidia.

Stereological analysis of transverse tubules and sarcoplasmic reticulum isolated from normal and dystrophic skeletal muscle

Vesicles isolated from the transverse tubules and sarcoplasmic reticulum of normal and dystrophic chicken skeletal muscle were analyzed for enzymatic activity and examined following freeze-fracture. A stereological procedure was used to determine particle density distributions on the resulting membrane fracture faces. The particle densities measured in this investigation were compared with those of an earlier study on intact muscle. Isolated sarcoplasmic reticulum vesicles showed a characteristically high P-face (cytoplasmic leaflet) particle density (5108 ± 169 particles/ μm 2) and a low E-face (luminal leaflet) particle density (505 ± 57 particles/ μm 2). Transverse tubule fractions showed a high E-face particle density (2346 ± 179 particles/ μ 2) as well as a substantial P-face particle density (1019 ± 129 particles/ μm 2). The high transverse tubule E-face particle density represents a characteristic morphological feature in the same way that the very high P-face particle density is characteristic of sarcoplasmic reticulum membranes. The major morphological alteration in dystrophic membranes was a shift in the E-face particle density distribution of isolated transverse tubules to a lower average particle density. (The E-face particle density of sarcoplasmic reticulum fractions showed no differences.)

The assembly of cellulose microfibrils in Valonia macrophysa Kütz

The assembly of cellulose microfibrils was investigated in artificially induced protoplasts of the alga, Valonia macrophysa (Siphonocladales). Primary-wall microfibrills, formed within 72 h of protoplast induction, are randomly oriented. Secondary-wall lamellae, which are produced within 96 h after protoplast induction, have more than three orientations of highly ordered microfibrils. The innermost, recently deposited micofibrils are not parallel with the cortical microtubules, thus indicating a more indirect role of microtubules in the orientation of microfibrils. Fine filamentous structures with a periodicity of 5.0-5.5 nm and the dimensions of actin were observed adjacent to the plasma membrane. Linear cellulose-terminal synthesizing complexes (TCs) consisting of three rows, each with 30-40 particles, were observed not only on the E fracture (EF) but also on P fracture (PF) faces of the plasma membrane. The TC appears to span both faces of the bimolecular leaflet. The average length of the TC is 350 nm, and the number of TCs per unit area during primary-wall synthesis is 1 per μm(2). Neither paired TCs nor granule bands characteristic of Oocystis were observed. Changes in TC structure and distribution during the conversion from primary- to secondary-wall formation have been described. Cellulose microfibril assembly in Valonia is discussed in relation to the process among other eukaryotic systems.

Ciliated and microvillous structures of rat olfactory and nasal respiratory epithelia. A study using ultra-rapid cryo-fixation followed by freeze-substitution or freeze-etching.

The olfactory epithelium of the Sprague-Dawley rat showed structures which indicate that freeze-substitution after ultra-rapid cryo-fixation is a better method for its preservation than conventional fixation techniques. A new feature is that matrices of the distal parts of olfactory cilia range in their staining intensity from very dense to electron-lucent. Outlines of structures are smooth and membrane features can be clearly seen. The textures of mucus from olfactory and respiratory epithelia are distinctly different after freeze-fracturing and deep-etching following cryo-fixation. Olfactory cilia show no microtubule-attached axonemal structures. Cross-sectional diameters are smaller after freeze-substitution than after freeze-fracturing. Intramembranous particle densities are lower in nine regions of three cell types in cryo-fixed olfactory and respiratory epithelia than in those chemically fixed and cryoprotected. The fracture faces of membranes from etched, cryo-fixed cells have holes, a result which probably accounts for differences in particle density between cryo-fixed and chemically-fixed, cryo-protected cells. Particle diameters are usually the same using both methods. Densities of intramembranous particles and particles plus holes are highest in supporting cell processes, followed by endings and cilia of olfactory receptor cells, and are lowest in respiratory cilia. Particle densities at outer and inner surfaces are higher than those in either fracture face. Outer surfaces show a good correlation from region to region with densities summated over both fracture faces.