Freeze-fracture studies on mitochondrial membranes of spermatocytes

Summary Separation of the two-folded lamina of the mitochondrial cristae occurs in mitochondria of spermatocytes and spermatids. Freeze-fracture exposes large areas of the inner and outer halves of the inner membrane. The surface of the outer half of the inner membrane is concave, with small numbers of intramembranous particles (IMPs). Its distinctive feature is the presence of protruding particles surrounding a pit. On the inner half of the inner membrane, there are large numbers of densely-packed, irregularly-distributed IMPs, among which regular pits are seen. Morphometric analysis and reconstructions suggest that these structures are channels in the mitochondrial membrane with an internal diameter of approximately 18 nm. It is uncertain whether such mitochondrial structures are confined to the spermatocyte or whether they may also occur in other cells.     

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

Summary 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.Supported by a research grant from the Medical Research Council of Canada (F.L.) (J.S.H.)     

Fine structure of the band 3 protein in human red cell membranes: Freeze-fracture studies

The major red cell membrane protein, band 3, is a glycoprotein which extends across the membrane from the extracellular space into the cytoplasmic compartment. It is widely held that band 3 is a component of the intramembrane particles (IMP) which can be demonstrated by freeze-fracture electron microscopy. In this study, we find that the outer surface poles of the IMP can be seen by freeze-etching after they are unmasked by proteolysis under conditions which excise the surrounding sialopeptides from the membrane. The poles appear as distinctive projections, 30–50 Å in diameter, the “ES particles.” The ES particles remain associated with the outer surface of the membrane following cleavage of the band 3 polypeptide by chymotrypsin or pronase. This is consistent with previous biochemical studies which have shown that the 38,000-dalton outer surface segment of band 3 is intercalated in the lipid bilayer. A granulofibrillar component at the inner surface of the membrane is provisonally identified as the 40,000-dalton inner-surface domain of band 3.     

Freeze-fracture electron microscopic observations on the effects of sulphydryl group reagents on human erythrocyte membranes

Freeze-fracture electron microscopy of human red blood cells at pH 7.4 and 5.5 reveals the presence of membrane elevations (50-100 nm diameter). These are also observed after incubation of the erythrocytes with N-ethylmaleimide but not after incubation with p-chloromercuribenzene sulphonate. Neither of the sulphydryl-group reagents affects the distribution or size of intramembrane particles. The findings are discussed in the light of the effects of mercurials on erythrocyte membrane proteins.     

Freeze-fracture study of the Drosophila photoreceptor membrane: mutations affecting membrane particle density

The photoreceptor membrane of Drosophila melanogaster (wild type, vitamin A-deprived wild type, and the mutants ninaAP228, ninaBP315, and oraJK84) was studied by freeze-fracture electron microscopy. The three mutations caused a decrease in the number of particles on the protoplasmic face of the rhabdomeric membrane. The ninaAP228 mutation affected only the peripheral photoreceptors (R1-6), while the ninaBP315 mutation affected both the peripheral (R1-6) and the central photoreceptors (R7). The oraJK84 mutation, which essentially eliminates R1-6 rhabdomeres, was found to drastically deplete the membrane particles in the vestigial R1-6 rhabdomeres but not in the normal rhabdomeres of R7 photoreceptors, suggesting that the failure of the oraJK84 mutant to form normal R1-6 rhabdomeres may be due to a defect in a major R1-6 photoreceptor-specific protein in the mutant. In all cases in which both the rhabdomeric particle density and rhodopsin content were studied, the mutations or vitamin A deprivation was found to reduce both these quantities, supporting the idea that at least the majority of the rhabdomeric membrane particles are closely associated with rhodopsin. Vitamin A deprivation and the mutations also reduced the number of particles in the plasma membrane as in the rhabdomeric membrane, suggesting that both classes of membrane contain rhodopsin.     

Freeze-fracture observations on membranes of dry and hydrated pollen from Collomia,Phoenix and Zea

Pollen from Collomia grandiflora Dougl. ex Lindl., Phoenix dactylifera L. and Zea mays L. was examined by freeze-fracture electron microscopy. Particular attention was paid to the organization of the cell membranes in the naturally dehydrated, as compared to the fully hydrated, state. All membranes examined had a normal bilayer organization similar to that seen in the hydrated cells of these and other plants. This organization of dry pollen membranes is discussed as it relates to physiological studies (e.g., leakage of ions during hydration), and to biophysical properties of biological and model membranes under various conditions of hydration and dehydration.Abbreviations EF, PF exoplasmic and protoplasmic fracture, respectively - H_II hexagonal II - IMPs intramembranous particles     

Fenestrated endothelium of the adrenal gland: Freeze-fracture studies

Little is known of how adrenal hormones pass from the interstitial to the vascular space. We have begun to examine the adrenal endothelium as a barrier to hormone passage, by the freeze-fracturing technique. The endothelium of both cortex and medulla is fenestrated. Fractures from both regions show endothelial cells to be extremely thin in regions where fenestrations are abundant. En face fractures show fenestrae disposed in tracts; the fenestrae reaching a distribution of 35/μ². In both cortex and medulla there are areas of continuous endothelium which contain caveolae. Structures believed to represent fenestra diaphragms contain randomly disposed particles and occasional pits. We have not identified in replicas the central ring and pore described in thin-sectioned material (Elfvin, 1965). The main differences between freeze-fractured aspects of cortical and medullary endothelium are the greater abundance of caveolae in the medulla and the size of the fenestrae (fenestra rims in the medulla are 525–780 Å in diameter; in the cortex 570–1660 Å). These differences may reflect the different embryological origins of the medulla and cortex. While caveolae may participate in hormone transport, there is no evidence for this. In the medulla the caveolae are more numerous and may have a function not necessarily related to transport. Possibly, caveolae play a role in processing hormones and related substances. For example, ATP and specific proteins are released as well as epinephrine during exocytosis from chromaffin cells. Epinephrine enters the vascular space but ATP does not. ATPase enzymes are a common feature of caveolae of other endothelia and may occur as well in adrenal endothelium.     

N-acyl phosphatidylethanolamines affect the lateral distribution of cholesterol in membranes

N-Acyl phosphatidylethanolamines are negatively charged phospholipids, which are naturally occurring albeit at low abundance. In this study, we have examined how the amide-linked acyl chain affected the membrane behavior of the N-acyl-1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (N-acyl-POPE) or N-acyl-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (N-acyl-DPPE), and how the molecules interacted with cholesterol. The gel-->liquid crystalline transition temperature of sonicated N-acyl phosphatidylethanolamine vesicles in water correlated positively with the number of palmitic acyl chains in the molecules. Based on diphenylhexatriene steady state anisotropy measurements, the presence of 33 mol% cholesterol in the membranes removed the phase transition from N-oleoyl-POPE bilayers, but failed to completely remove it from N-palmitoyl-DPPE and N-palmitoyl-POPE bilayers, suggesting rather weak interaction of cholesterol with the N-saturated NAPEs. The rate of cholesterol desorption from mixed monolayers containing N-palmitoyl-DPPE and cholesterol (1:1 molar ratio) was much higher compared to cholesterol/DPPE binary monolayers, suggesting a weak cholesterol interaction with N-palmitoyl-DPPE also in monolayers. In bilayer membranes, both N-palmitoyl-POPE and N-palmitoyl-DPPE failed to form sterol-rich domains, and in fact appeared to displace sterol from sterol/N-palmitoyl-sphingomyelin domains. The present data provide new information about the effects of saturated NAPEs on the lateral distribution of cholesterol in NAPE-containing membranes. These findings may be of relevance to neural cells which accumulate NAPEs during stress and cell injury.     

Cell cholesterol efflux: integration of old and new observations provides new insights

Numerous studies using a variety of cell/acceptor combinations have demonstrated differences in cholesterol efflux among cells. These studies also show that different acceptors, ranging from simple molecules like cyclodextrins to serum, stimulate efflux through a variety of mechanisms. By combining early observations with data derived from recent studies, it is now possible to formulate a model for cell cholesterol efflux which proposes that an array of different mechanisms, including aqueous diffusion, lipid-free apolipoprotein membrane microsolubilization, and SR-BI-mediated cholesterol exchange contribute to cholesterol flux. In this model the relative importance of each mechanism would be determined both by the cell type and the nature of the extracellular cholesterol acceptor.     

Freeze-fracture studies of the thecal membranes of Gonyaulax polyedra: circadian changes in the particles of one membrane face.

Intramembrane faces were visualized in the marine dinoflagellate Gonyaulax polyedra by the freeze-fracture technique, in order to test a prediction of a membrane model for circadian oscillations--i.e;, that membrane particle distribution and size change with time in the circadian cycle. Cells from each of four cell suspensions in continuous light (500 1x, 20-21 degrees C) were frozen, without fixation or cryoprotection, at four circadian times in a cycle. This paper reports findings concerning the membranes associated with the theca, particularly the cytoplasmic membrane and the membrane of the large peripheral vesicle. While the number and size distribution of the particles of the PF face of the cytoplasmic membrane were constant with time, those of the EF face of the peripheral vesicle doubled in number at 18 h circadian time as compared with 06 h. Particles of the 120-A size class, in particular, were more numerous at 12 and 18 h circadian time than at 00 and 06 h. While the finding does not provide definitive confirmation of the membrane hypothesis for circadian rhythms, it is consistent with this model. It is suggested that the peripheral vesicle may be the site of bioluminescence in Gonyaulax.     

Influence of cholesterol on electroporation of bilayer lipid membranes: chronopotentiometric studies

This paper presents the results of constant-current (chronopotentiometric) measurements of the egg yolk phosphatidylcholine (PC) bilayer membrane without and with cholesterol. The experiments were performed on planar bilayer lipid membrane (BLM) formed by the Mueller-Rudin method. It is demonstrated that the constant-intensity current flow through bilayer membranes generated fluctuating pores in their structure. The presence of cholesterol in the membrane caused an increase in the value of the breakdown potential. It is postulated that greater stability of the bilayer with cholesterol can result from an increased critical pore radius (at which the bilayer would undergo irreversible rupture). This confirms that cholesterol has a stabilizing effect on BLM. Besides, our results suggest that addition of cholesterol causes shift in the distribution of pore conductance towards a smaller value. It is suggested that this can be connected with the phenomenon of domain formation in the membranes containing high concentration of cholesterol. Moreover, it is shown that chronopotentiometry with programmable current intensity is a promising method for observation of the membrane recovery process.     

Influence of phospholipid unsaturation on the cholesterol distribution in membranes.

Over the last half decade, we have studied saturated and unsaturated phosphatidylcholine (PC)-cholesterol membranes, with special attention paid to fluid-phase immiscibility in cis-unsaturated PC-cholesterol membranes. The investigations were carried out with fatty acid and sterol analogue spin labels for which reorientational diffusion of the nitroxide was measured using conventional ESR technique. We also used saturation recovery ESR technique where dual probes were utilized. Bimolecular collision rates between a membrane-soluble square-planar copper complex,3-ethoxy-2-oxobutyraldehyde bis(N4,N4-dimethylthiosemicarbazonato)copper(II) (CuKTMS2) and one of several nitroxide radical lipid-type spin labels were determined by measuring the nitroxide spin-lattice relaxation time (T1). The results obtained in all these studies can be explained if the following model is assumed: 1) at physiological temperatures, fluid-phase micro-immiscibility takes place in cis-unsaturated PC-cholesterol membranes, which induces cholesterol-rich domains in the membrane due to the steric nonconformability between the rigid fused-ring structure of cholesterol and the 30 degrees bend at the cis double bond of the alkyl chains of unsaturated PC. 2) The cholesterol-rich domains are small and/or of short lifetime (10(-9) s to less than 10(-7) s). Our results also suggest that the extra space that is available for conformational disorder and accommodation of small molecules is created in the central part of the bilayer by intercalation of cholesterol in cis-unsaturated PC membrane due to the mismatch in the hydrophobic length and nonconformability between cis-unsaturated PC alkyl chains and the bulky tetracyclic ring of cholesterol.     

Influence of cholesterol on electroporation of bilayer lipid membranes: chronopotentiometric studies

This paper presents the results of constant-current (chronopotentiometric) measurements of the egg yolk phosphatidylcholine (PC) bilayer membrane without and with cholesterol. The experiments were performed on planar bilayer lipid membrane (BLM) formed by the Mueller-Rudin method. It is demonstrated that the constant-intensity current flow through bilayer membranes generated fluctuating pores in their structure. The presence of cholesterol in the membrane caused an increase in the value of the breakdown potential. It is postulated that greater stability of the bilayer with cholesterol can result from an increased critical pore radius (at which the bilayer would undergo irreversible rupture). This confirms that cholesterol has a stabilizing effect on BLM. Besides, our results suggest that addition of cholesterol causes shift in the distribution of pore conductance towards a smaller value. It is suggested that this can be connected with the phenomenon of domain formation in the membranes containing high concentration of cholesterol. Moreover, it is shown that chronopotentiometry with programmable current intensity is a promising method for observation of the membrane recovery process.     

Fluorescent cholesterol derivatives in studies of membranes and lipoproteins

The pathways of fluorescent chromophores introduction into the cholesterol molecule are reviewed and physicochemical, optical properties and biological identity of fluorescent cholesterol derivatives (FCD) to the natural analogues are briefly characterized. Methods of the FCD incorporation into biological objects are discussed. The main part of the review is devoted to the use of FCD to investigate the model and biological membranes, blood plasma lipoproteins and their interactions with cells. The first attempts of the FCD application in the study of sterol-carrier proteins are discussed.     

NBD-cholesterol probes to track cholesterol distribution in model membranes

A series of cholesterol (Chol) probes with NBD and Dansyl fluorophores attached to the 3-hydroxyl position via carbamate linkers has been designed and synthesized and their ability to mimic the behavior of natural cholesterol in bilayer membranes has been examined. Fluorescence spectroscopy data indicate that the NBD-labeled lipids are located in the polar headgroup region of the bilayer with their position varying with the method of fluorophore attachment and the linker length. The partitioning of the Chol probes between liquid-ordered (L_o) and liquid-disordered (L_o) phases in supported bilayers prepared from ternary lipid mixtures of DOPC, Chol and either egg sphingomyelin or DPPC was examined by fluorescence microscopy. The carbamate-linked NBD-Chols show a stronger preference for partitioning into L_o domains than does a structurally similar probe with an ester linkage, indicating the importance of careful optimization of probe and linker to provide the best Chol mimic. Comparison of the partitioning of NBD probes to literature data for native Chol indicates that the probes reproduce well the modest enrichment of Chol in L_o domains as well as the ceramide-induced displacement of Chol. One NBD probe was used to follow the dynamic redistribution of Chol in phase separated membranes in response to in situ ceramide generation. This provides the first direct optical visualization of Chol redistribution during enzymatic ceramide generation and allows the assignment of new bilayer regions that exclude dye and have high lateral adhesion to ceramide-rich regions.     

Freeze-fracture studies on barley plastid membranes. VI. Location of the P700-chlorophyll a-protein 1

The photosystem I mutant of barley, viridis-zb63, which totally lacks the P700-chlorophyll a-protein 1 was characterised by rotary shadowed, freeze-fracture electron microscopy. Objective measurements were made of particle density and size distribution for all four fracture faces, and compared with values for wild-type. A highly significant reduction in the size of the PFu particles was found, which could be attributed to the loss of a population of large particles from the mutant PFu face. A corresponding loss of EFu pits was also observed. It is concluded that the photosystem I reaction centre and two or three ancillary polypeptides are located in the large PFu particles which account for two-thirds of the total, and that these particles span the membrane. Since no differences were seen on the PFs and EFs faces, there was no evidence for the localisation of any photosystem I in appressed granal membranes, supporting the concept of extreme lateral heterogeneity. A model is presented of the localisation of different functional polypeptide units to different freeze-fracture particles within the membrane. A peculiar feature of viridis-zb63 thylakoids was the presence of EFs particle arrays in vivo.     

Freeze-fracture studies in the brown algaAsteronema rhodochortonoides

Summary The gross structure of the cell wall and the organization of the plasmalemma of the filamentous brown algaAsteronema rhodochortonoides were examined in replicas of freeze-fractured cells. The protoplasmic fracture face (PF) of the plasmalemma, apart from the single particles, exhibits two particular particle complexes, i.e., single linear arrays of closely packed particles, and well defined particle pentads. The former display a consistent relationship with the ends of microfibril imprints and therefore are considered as terminal complexes (TCs). They seem to be composed of subunits, each one consisting of two particles. The average diameter of the particles is 7 nm. The number of the subunits forming the TCs varies between 2 and 40. Short TCs, consisting of 3–5 subunits were also found on the PF of dictyosome vesicles, a fact suggesting the involvement of the Golgi apparatus in exocytosis of preformed TC portions. The occurrence, distribution and size of the TCs appear to be related to the developmental stage of the cell. A large number of TCs occur in actively growing cells, while a few or no TCs are found in differentiated cells. The pentads are rectangular structures consisting of five particles, four in the corners and one in the centre. Their dimensions are very constant, but their occurrence and distribution varies. They occur in young developing cells where TCs are few or absent, but were also observed in areas showing many TCs. In differentiated cells no pentads were found. Pentad-like structures were rarely observed on the PF of dictyosome vesicles or cisternae. The observations support the hypothesis that pentads are involved in the synthesis of matrix polysaccharides, which are the major components of brown algal cell wall and their synthesis begins before that of cellulose.Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement