Effects of lipid-phase separation on the filipin action on membranes of ergosterol-replaced Tetrahymena cells,as studied by freeze-fracture electron microscopy

The effects of lipid-phase separation on the filipin action on pellicle membranes of ergosterol-replaced Tetrahymena pyriformis cells were studied by freeze-fracture electron microscopy. The pellicle membranes with phase separations induced by chilling from 34°C (growth temperature) to lower temperatures (30, 22 and 15°C) were treated with filipin. This produced filipin-induced lesions (“pits”) only in the particulated (liquid) regions along the margin between solid and liquid domains, while they were produced in the particle-free (solid) areas when membranes were chilled to 15°C. The pellicle membranes with lesions induced by filipin at 34°C were chilled to 22°C. This chilling raised larger particle-free areas and more condensed particle-aggregations on the membranes than on the membranes without the filipin treatment. These results suggest that the membrane fluidity affects induction and development of the ergosterol-filipin complex in the membrane.     

Possible association of segregated lipid domains of Mycoplasma gallisepticum membranes with cell resistance to osmotic lysis.

Freeze-fracturing of cholesterol-rich Mycoplasma gallisepticum membranes from cells grown in a medium containing horse serum revealed particle-free patches. The patches appeared in cells quenched from either 4 or 37 degrees C. Particle-free patches also occurred in membranes of cells grown in a serum-free medium supplemented with egg-phosphatidylcholine but not in membranes of cells grown with dioleoylphosphatidylcholine. The appearance of particle-free patches was attributed to the presence of disaturated phosphatidylcholine (PC) molecules in M. gallisepticum membranes, which were synthesized by the insertion of a saturated fatty acid at position 2 of lysophosphatidylcholine derived from exogenous PC present in the growth medium. Consequences of the synthesis of the disaturated PC also included a decrease in osmotic fragility and the ability of the cells to be permeated by K+. Electron paramagnetic resonance and fluorescence polarization measurements revealed that the fluidity of the lipid domain in the protein-rich M. gallisepticum membranes was almost identical to that of an aqueous dispersion of M. gallisepticum membrane lipids. Furthermore, the electron paramagnetic resonance spectra of the membranes were single-component spectra showing no indication of immobilized regions. The possibility that the osmotic resistance of M. gallisepticum cells is associated with the particle-free patches rather than with a restricted membrane fluidity caused by membrane proteins is discussed.     

Freeze-fracture evidence for the presence of cholesterol in particle- free patches of basal disks and the plasma membrane of retinal rod outer segments of mice and frogs

The freeze-fracture technique was used to examine the membranes of the photoreceptors of mice and frogs. Particle-free patches were found in the plasma membrane and basal disk membranes of the outer segments of both mice and frogs housed at room temperature, but not in frogs kept in a cold room. These patches were shown not to be artifacts of cryoprotection or fixation, and they persisted when fresh isolated outer segments were frozen by an ultrarapid method. They were also found to persist in mouse rods when retinas were incubated and subsequently fixed at temperatures up to 80 degrees C. Cholesterol was implicated as a significant component of the patches by the observation that, in the outer segments, pits, induced by treatment with the sterol-specific polyene antibiotic filipin, were present in and confined to the particle-free patches. That these lesions are not inherently limited to particle-free membrane areas was evident in the apical plasma membrane of the photoreceptor inner segments, where particles and pits were intermixed. Treatment with saponin, a surface-active agent which specifically complexes cholesterol, resulted in the disappearance of the particle-free patches. Patches were found in basal disks of both mouse and frog rods but not in older disks nearer the pigment epithelium, which indicates that changes occur in the composition of disk membranes and/or in the molecular ordering of their protein and lipid components during the early phase of their transit from the base towards the apex of the outer segment.     

Freeze-fracture of sperm of Plumbago zeylanica L. in pollen and in vitro

 Sperm of Plumbago zeylanica are dimorphic with regard to numbers of mitochondria and plastids. In most cases examined, the plastid-rich sperm fused with the egg while the sperm with fewer plastids fused with the central cell. However, plastids cannot be directly responsible for fusion because fusion occurs between the plasma membranes of egg and sperm. The question is whether sperm cell membranes are distinctive and possibly dimorphic. Sperm in whole pollen grains and isolated sperm were freeze-fractured. In pollen, freeze-fractured sperm appeared only in cross fractures. No extended membrane fracture faces of sperm were found. Among isolated sperm, two sizes of sperm with different organelles were observed. Isolated sperm were assigned to two categories based on cell diameter and on size and density of organelles. Membrane particles on most sperm were arranged without distinctive pattern. Some hexagonal arrays were observed. In sperm that had been maintained at 4°C, particle-free areas, a probable consequence of lipid phase separations, appeared on plasma membrane fracture faces. No unique fracture patterns and no patterns of dimorphism were detected on freeze-fractured plasma membranes of Plumbago sperm. Received: 14 January 1997 / Revision accepted: 6 June 1997     

Freeze-fracture evidence of gel-phase lipid in membranes of senescing cowpea cotyledons

The structural details of membrane organization in germinating and senescing cotyledons of cowpea (Vigna unguiculata (L.) Walp.) were studied by thin section and freeze-fracture electron microscopy. Germination- and senescence-related changes in the ultrastructure of parenchymal cells of cowpea cotyledons, as detected in thin sections, closely resemble those described for other leguminous seeds. Additionally, electron-dense deposits associated with the membranes, particularly the plasmalemma and endoplasmic reticulum, were seen to increase with advancing senescence. Freeze-fracture electron microscopy demonstrated that the membranes of cotyledons of 2-d-old seedings appear to be normal, with evenly dispersed intramembranous particles. However by 4 d, small areas or domains of the plasmalemma were free of intramembranous particles. These particle-free areas increased in both size and number as senescence progressed. We interpret these particle-free areas to be structural evidence for lateral phase separations of the membrane lipids into microdomains of gel-phase lipid from which intrinsic membrane proteins are excluded. Our results support wide-angle X-ray diffraction studies which have demonstrated the presence of gel-phase lipids in senescing bean cotyledons.Abbreviations EF exoplasmic fracture - ER endoplasmic reticulum - ESR electron-spin resonance - IMP(s) intramembranous particle(s) - PF protoplasmic fracture     

Effects of lipid-phase separation on the filipin action on membranes of ergosterol-replaced Tetrahymena cells, as studied by freeze-fracture electron microscopy.

The effects of lipid-phase separation on the filipin action on pellicle membranes of ergosterol-replaced Tetrahymena pyriformis cells were studied by freeze-fracture electron microscopy. The pellicle membranes with phase separations induced by chilling from 34 degrees C (growth temperature) to lower temperatures (30, 22 and 15 degrees C) were treated with filipin. This produced filipin-induced lesions ("pits") only in the particulated (liquid) regions along the margin between solid and liquid domains, while they were produced in the particle-free (solid) areas when membranes were chilled to 15 degrees C. The pellicle membranes with lesions induced by filipin at 34 degrees C were chilled to 22 degrees C. This chilling raised larger particle-free areas and more condensed particle-aggregations on the membranes than on the membranes without the filipin treatment. These results suggest that the membrane fluidity affects induction and development of the ergosterol-filipin complex in the membrane.     

Temperature-induced transitions of porcine intestinal brush border membranes

Thermotropic transitions of the membrane components in porcine intestinal brush border membranes were studied by means of fluorimetry using a fluorogenic thiol reagent, N-[7-dimethylamino-4-methylcoumarinyl]maleimide (DACM), and a lipophilic fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH). 1. The reactivity of the sulfhydryl groups of the membrane proteins with DACM was dependent on temperature, with a transition point at about 33°C. A conspicuous transition was also observed in the relation between temperature and the fluorescence intensity of DACM-labeled membranes at 35°C. 2. Temperature dependence profiles of the solubilization of DPH in the membranes and of the fluorescence polarization of DPH-membrane complex suggested that the phase transition of the lipid from gel to liquid-crystalline state occurs over a temperature range of 30 to 35°C. 3. Efficient fluorescence energy transfer was observed from tryptophan residues of the membrane proteins to DPH located in the lipid phase of the membranes, and its efficiency was extremely enhanced, dependent on temperature, above 35°C. The intensity of the tryptophan fluorescence of the membrane proteins decreased with increasing temperature and a discontinuity was observed at about 33°C. Based on these results, it may be concluded that there are co-operative interactions between proteins and lipids in the membranes and that the temperature-induced conformational changes of the membrane proteins are closely related to the dynamics of the hydrocarbon cores of the lipid.     

Freeze-fracture of membrane fusions during exocytosis in pancreatic B- cells

To examine the freeze-fracture appearance of membrane alterations at sites of exocytosis in mammalian cells, we studied the secretory granule and plasma membrane of rat pancreatic B-cells during glucose-stimulated insulin secretion. Constant features observed were the scarcity of particles in secretory-granule P-fracture faces and the almost total clearance of intramembranous particles in P-and E fracture faces of the plasma membrane in areas of close apposition of these two membranes preceding fusion; also observed was the temporary persistence of particle-cleared regions after the fusion was completed. Our observations thus support the concept that membranes fuse at sites of closely apposed, particle-free regions and that the physiologically created clear areas found in freeze-fracture replicas of the plasma membrane are the hallmarks of incipient or recent membrane fusion.     

Studies on Tetrahymena membranes. Palmitoyl-coenzyme a desaturase, a possible key enzyme for temperature adaptation in Tetrahymena microsomes.

(1) Microsomes from a thermotolerant Tetrahymena NT-1 catalyze the conversion of palmitoyl-CoA to palmitoleate. (2) Palmitoyl-CoA desaturase enzyme requires molecular oxygen and NADH or NADPH as cofactor and its activity is inhibited by cyanide. A pH optimum range 7.0--7.3 is observed. (3) There is a clear break at 30 degrees C and a slight bend around 15 degrees C in the Arrhenius plots of palmitoyl-CoA desaturase activity. (4) After quenching from 39.5 degrees C, at 26 degrees C microsomal membranes show small particle-free areas, when examined by freeze-fracture electron microscopy, indicating the onset of phase separation. Larger smooth areas devoid of membrane-intercalated particles are observed in microsomes at 23 and 15 degrees C. The results support evidence that the thermally induced transition of desaturase enzyme activity in related to the altered membrane properties due to temperature change.     

Heat shock temperature acclimation of normal secretory protein synthesis in barley aleurone cells

Exposure of barley (Hordeum vulgare L. cv. Himalaya) aleurone layers to 40°C for a period of 3 h results in the selective suppression of the synthesis and secretion of hydrolytic enzymes; other normal cellular protein synthesis continues during heat shock. This suppression is correlated with secretory protein mRNA destabilization and the dissociation of stacked ER lamellae during heat shock (Belanger et al. 1986, Proceedings of the National Academy of Sciences USA 83, pp. 1354–1358). In this report we examined the effect of exposure to extended periods of heat shock. If exposure to 40°C was continued for a period of 18 h, the synthesis of α-amylase, the predominant secreted hydrolase, resumed. This was accompanied by increased α-amylase mRNA levels and the reformation of ER lamellae. Though initial exposure (3 h) to 40°C reduced protein secretion to ～10% of that observed in aleurone cells maintained at 25°C, exposure for prolonged periods (16–20 h) permitted the resumption of protein secretion to ～66% of non-heat-shocked control levels. The resumption of normal secretory protein synthesis during prolonged exposure to 40°C was correlated with an increase in the incorporation of [¹⁴C]glycerol into phosphatidylcholine and an increase in the ratio of saturated to unsaturated fatty acids in lipids isolated from ER membrane preparations. Increased fatty acid saturation has been demonstrated to enhance thermostability in biological membranes, and such changes in membrane composition may be important to the recovery of secretory protein synthesis at the ER.     

Resistance of the neck plasma membrane between the mother and the bud of Saccharomyces cerevisiae and Candida albicans to amphotericin B-induced deformation

Abstract The polyene antibiotic, amphotericin B, at high concentrations (5–20 μg/ml) induced particle-free smooth areas in the plasma membranes of Saccharomyces cerevisiae and Candida albicans . These areas occured more or less over the entire plasma membrane of unbudded cells. In budded cells, however, the neck between the mother and bud did not undergo deformation. This suggests the strong interaction between the filamentous ring, which is firmly attached to the neck plasma membrane, and plasma membrane particles in the neck regions.     

Effect of temperature on glycerol metabolism in membranes and on phospholipases C and D of germinating pea embryos

The effect of temperature of imbibition on the synthesis and turnover of membrane phosphatidyl choline was studied. Pea seeds (Pisum sativum cv. Alaska) were imbibed in [U-¹⁴C]glycerol and then germinated. Seeds were kept constantly either at 5° or 25°, or were imbibed at one temperature and then germinated at the other one. Glycerol incorporation into phosphatidyl choline in the ER and the plasma membrane, obtained from the embryonic axes after germination, and the glycerol pool were measured. Embryos from seeds kept constantly at 25° showed a rapid incorporation of glycerol into membranes followed by a loss of label; in embryos from seeds kept at 5° incorporation was much lower. Embryos from seeds transferred from 25° to 5° behaved as if continuously kept at 25°, while the behaviour of the embryos from seeds transferred from 5° to 25° resembled embryos from seeds maintained at 5°. The glycerol content of the axes rose during imbibition and fell thereafter. The activities of phospholipases C and D also responded to the initial temperature of imbibition, but the two activities changed differently. The results are discussed in relation to the effect of transient exposure to temperature changes in the seed membranes and the possible way in which such changes are sensed.     

Temperature dependence of the photosynthetic activities in the thylakoid membranes from the blue-green alga Anacystis nidulans

The photosynthetic electron transport and phosphorylation reactions were measured in the room temperature region in the thylakoid membranes prepared from the blue-green alga, Anacystis nidulans. The Arrhenius plot of the Hill reaction with 2,6-dichlorophenolindophenol showed a distinct break of straight lines at 21°C in the membranes from cells grown at 38°C, and at 12°C in those from cells grown at 28°C. The Arrhenius plot of the Hill reaction with ferricyanide showed a break at 13°C in the membranes from cells grown at 38°C, and at 7°C in those from cells grown at 28°C. On the other hand, the Arrhenius plot of the System I reaction with methylviologen as an electron acceptor and 2,6-dichlorophenolindophenol and ascorbate as an electron donor system was composed of a straight line in the membranes from cells grown at 28°C as well as at 38°C. The Arrhenius plot of the System II reaction measured by the ferricyanide reduction mediated by silicotungstate in the presence of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea also showed a break at 11°C in the membranes from cells grown at 38°C.The Arrhenius plot of the phosphorylation mediated by N-methylphenazonium methylsulfate showed a break at 21°C in the membranes from cells grown at 38°C and at 12°C in those from cells grown at 28°C. The Arrhenius plot of the phosphorylation mediated by the System I reaction showed a break at 24°C in the membranes from cells grown at 38°C.The characteristic features in the Arrhenius plots of the photosynthetic electron transport and phosphorylation reactions are discussed in terms of the transition of physical phase of the thylakoid membrane lipids.     

Demonstration of 5′-nucleotidase activity in unfixed cryostat sections of rat liver using a combined light- and electron-microscope procedure

Summary In the present study a technique was developed to demonstrate 5′-nucleotidase activity in unfixed cryostat sections of rat liver at the light- and electron-microscope level using a semipermeable membrane. In order to retain the ultrastructure of the unfixed material as much as possible, incubations were also performed at 4°C rather than at 37°C. The optimized incubation medium contained 300 mm Tris-maleate buffer, pH 7.2, 5 mm adenosine monophosphate as substrate, 30 mm cerium chloride as capturing agent for liberated phosphate, 10 mm magnesium chloride as activator and 1.5% agar. At the light-microscope level, similar localizations of 5′-nucleotidase activity were obtained when incubations were performed at 37°C and 4°C. Enzyme activity was present mainly at bile canalicular membranes and at sinusoidal membranes of hepatocytes; total activity was higher in pericentral than in periportal areas. Cytophotometric analyses revealed that specific formation of final reaction product (FRP) (test minus control reaction) at 37°C followed a hyperbolic curve with time. A linear relationship was found between specific amounts of FRP and section thickness up to 8μm. 5′-Nucleotidase activity was about three-fold higher after incubation for 30 min at 37°C than at 4°C. At the electron-microscope level, it was demonstrated that the ultrastructure of rat liver was rather well-preserved after incubating unfixed cryostat sections attached to a semipermeable membrane and electron-dense FRP was found at bile canalicular and sinusoidal plasma membrane of hepatocytes. The most distinct changes in ultrastructure after incubation at 37°C, in comparison with that at 4°C, were the appearance of multi-lamellar structures at bile canaliculi at 37°C. We conclude that the present method is valid for the demonstration of 5′-nucleotidase activity in unfixed cryostat sections of rat liver at both the light- and electron-microscope levels and that hypothermic incubations improve ultrastructural morphology substantially.     

Membrane events involved in myoblast fusion

Myoblast fusion has been studied in cultures of chick embryonic muscle utilizing ultrastructural techniques. The multinucleated muscle cells (myotubes) are generated by the fusion of two plasma membranes from adjacent cells, apparently by forming a single bilayer that is particle-free in freeze-fracture replicas. This single bilayer subsequently collapses, and cytoplasmic continuity is established between the cells. The fusion between the two plasma membranes appears to take place primarily within particle-free domains (probably phospholipid enriched), and cytoplasmic unilamellar, particle-free vesicles are occasionally associated with these regions. These vesicles structurally resemble phospholipid vesicles (liposomes). They are present in normal myoblasts, but they are absent in certain fusion-arrested myoblast popluations, such as those treated with either 5-bromo-deoxyuridine (BUdR), cycloheximide (CHX), or pospholipase C (PLC). The unilamellar, particle-free vesicles are present in close proximity to the plasma membranes, and physical contact is observed frequently between the vesicle membrane and the plasma membrane. The regions of vesicle membrane-plasma membrane interaction are characteristically free of intramembrane particles. A model for myoblast fusion is presented that is based onan interpretation of these observations. This model suggests that the cytoplasmic vesicles initiate the generation of particle-depleted membrane domains, both being essential components in the fusion process.     

Effects of semen preservation on boar spermatozoa head membranes

Head plasma membranes were isolated from the sperm-rich fraction of boar semen and from sperm-rich semen that had been subjected to three commercial preservation processes: Ex tended for fresh insemination (extended), prepared for freezing but not frozen (cooled), and stored frozen for 3-5 weeks (frozen-thawed). Fluorescence polarization was used to determine fluidity of the membranes of all samples for 160 min at 25°C and also for membranes from the sperm-rich and extended semen during cooling and reheating (25 to 5 to 40°C, 0.4°C/min). Head plasma membranes from extended semen were initially more fluid than from other sources (P < 0.05). Fluidity of head membranes from all sources decreased at 25°C, but the rate of decrease was significantly lower for membranes from cooled and lower again for membranes from frozen-thawed semen. Cooling to 5°C reduced the rate of fluidity change for plasma membranes from the spernvrich fraction, while heating over 30°C caused a signifi cantly greater decrease. The presence of Ca⁺⁺ (10 mM) lowered the fluidity of the head plasma membranes from sperm-rich and extended semen over time at 25°C but did not affect the membranes from the cooled or frozen-thawed semen. The change in head plasma membrane fluidity at 25°C may reflect the dynamic nature of spermatozoa membranes prior to fertilization. Extenders, preservation processes and temperature changes have a strong influence on head plasma membrane fluidity and therefore the molecular organization of this membrane.     

A spin label study of E. coli membrane vesicles

The phase transition in E. coli membrane vesicles has been investigated by the spin labeling technique. N-oxyl-4′,4′-dimethyloxazolidine derivatives of stearic acid were incorporated into the vesicles. The results suggest that there are two phase transitions in these bacterial membrane vesicles (one at ≈20°C and the other at ≈30°C). These two phase transitions may be related to some of the functional properties of the membranes.     

Freeze-fracture images of exocytosis and endocytosis in anterior pituitary cells of rabbits and mice

Summary Freeze-fracture images of exocytosis and endocytosis were studied in various kinds of secretory cells of the anterior pituitary of mice and rabbits. Exocytotic figures are frequently observed in thin section of the anterior pituitary cells. In freeze-fracture images, small elevated membrane areas without membrane particles are often seen on the PF of the plasma membrane of the secretory cells. There is a secretory granule in the cytoplasm just beneath the particle-free membrane area, and limiting membrane of the granule is also devoid of the membrane particles at the part facing the plasma membrane. The fusion of membranes for exocytosis may occur at this particle-free area.The limiting membrane of the granule which is continuous with the plasma membrane is almost always coated after release of the granule core. This invagination of coated membrane may be an initiation site for the membrane retrieval after exocytosis. In freeze-fracture images, this depressed region with an accumulation of the membrane particles is observed on the PF of the plasma membrane. This particle-rich depressed region is thought to correspond to the coated area of the plasma membrane observed in thin section. It is thought that the membrane retrieval by pinocytosis initiates at the particle-rich depressed region of the plasma membrane.This study was supported by a grant from the Japan Ministry of Education     

Turnover of protein components of the plasma membrane of Saccharomyces cerevisiae

The peptide composition of plasma membrane in Saccharomyces cerevisiae cells growing at different temperatures between 18 and 38°C was studied using SDS-polyacrylamide gel electrophoresis. Stability of the proteins, both qualitative and quantitative, was observed at the tested temperatures. Treatment for 2 h with cycloheximide decreased by about 50% the amount of a 80 kDa membrane peptide at 18, 23, 28 and 33°C, with no other apparent effects. At 38°C the 80 kDa peptide was not affected by the presence of the drug. Addition of tunicamycin to cultures at concentrations partially inhibitory to growth caused a large accumulation of the 80 kDa peptide in the plasma membrane, which cycloheximide did not modify. Pulse-chase experiments indicated a low rate of turnover of total plasma membranes in cells growing at 18 and 28°C. In contrast, at 38°C about 50% of the radioactivity in plasma membranes disappeared after a 2 h chase. The 80 kDa protein band was the only one with significant differential decay.     

Pressure dependence of pyrene excimer fluorescence in human erythrocyte membranes

The intensity of pyrene excimer fluorescence in human erythrocyte membranes and in sonicated dispersions of the membrane lipid (liposomes) was examined as a function of pressure (1–2080 bar) and temperature (5–40°C). Higher pressure or lower temperature decreased the excimer/monomer intensity ratios. A thermotropic transition was detected in both membranes and liposomes by plots of the logarithm of the excimer/monomer intensity ratio versus 1/K. The transition temperature of the membranes was 19–21°C at 1 bar and 28–31°C at 450 bar, a shift with pressure of approx. 20–22 K per kbar. Corresponding transition temperatures of the liposomes were 21°C at 1 bar and 33°C at 450 bar, a shift of approx. 27 K per kbar. The observed pressure dependence of the thermotropic transition temperature is similar to that reported for phospholipid bilayers and greatly exceeds that of protein conformation changes. In concert with the liposome studies the results provide direct evidence for a lipid transition in the erythrocyte membrane.