Improved antigen retrieval in freeze-fracture cytochemistry by evaporation of carbon as first replication layer

The recently developed freeze-fracture replica immunolabeling technique uses sodium dodecyl sulfate to clean replicas obtained from chemically unfixed, rapidly frozen cells by evaporation of platinum as first and carbon as second replication layer. The detergent dissolves remains of cellular material with the exception of components which are in direct contact to the replica film. Membrane lipids and membrane protein complexes of the protoplasmic and the exoplasmic membrane halves remain attached to the replica film and are accessible for cytochemical localization. We immunolabeled the membrane proteins caveolin-1 and connexin 43 in mouse cell lines as well as the membrane attached protein tetrachloroethene reductive dehalogenase (PceA) in bacterial cells at freeze-fracture replicas generated by different evaporation parameters. The labeling experiments for caveolin-1 and the PceA showed that freeze-fracture replication of cellular membranes accomplished with thin platinum layers as well as replication with carbon as first evaporation layer lead in these cases to an improved antigen retrieval, whereas the labeling efficiency of connexin 43 was not affected by different evaporation conditions.     

SDS-digested freeze-fracture replica labeling electron microscopy to study the two-dimensional distribution of integral membrane proteins and phospholipids in biomembranes: practical procedure, interpretation and application

 Recently, we have developed a quick-freezing/freeze-fracture replica labeling technique, sodium dodecyl sulfate (SDS)-digested freeze-fracture replica labeling (SDS-FRL), to study the two-dimensional distribution of cytochemical labeling on the membrane surface and the relationship of this distribution to images of freeze-fracture replicas created by platinum shadowing. In SDS-FRL, unfixed, quick-frozen cells, after freeze-fracture and platinum/carbon shadowing, are treated with SDS. The detergent dissolves unfractured areas of the cell membranes, with the release of the cytoplasmic contents. The cytoplasmic and exoplasmic membrane surfaces can be then labeled cytochemically. Integral membrane proteins, revealed as intramembrane particles by freeze-fracture replication, which are indistinguishable on a purely morphological basis, can be selectively labeled by SDS-FRL with specific antibody. In addition, this approach can be applied to examine the transmembrane phospholipid distribution in various cell and intracellular membranes. In this review, we describe the practical procedure for SDS-FRL in detail, present its application to labeling of various membrane components, and briefly discuss the possibility of a combination of SDS-FRL with atomic force microscopy. Accepted: 1 November 1996     

High-resolution immunocytochemical labeling of replicas with ultrasmall gold.

The use of 10-15-nm gold probes in freeze-fracture immunocytochemistry sometimes results in poor immunogold labeling. Replica sites are labeled with only one or two gold particles, making it unlikely that the labeling depicts the true distribution of antigen. In this study, the feasibility of using ultrasmall ( approximately 1.4-nm) gold probes for immunocytochemical labeling of replicas was examined. When HLA Class I in neutrophil membrane replicas was labeled with various sized immunogold particles as the secondary detection system, the apparent distribution density was inversely related to the size of the particles (1.4-nm > 5-nm >10-nm >15-nm). Indeed, the density of the apparent distribution of HLA Class I labeled with 1.4-nm gold particles was about sevenfold greater than when labeling was carried out with the 10-nm gold particles. Similar results were obtained with CD16, another neutrophil membrane protein. Silver enhancement was required to visualize the 1.4-nm gold particles, but this procedure did not adversely affect replica membranes. These results suggest that, when followed by silver enhancement, 1.4-nm gold particles are effective probes for achieving high-resolution immunocytochemical labeling of replicas.(J Histochem Cytochem 47:569-573, 1999)     

Stereo views and immunogold labeling of the pellicular microtubules at the inner surface of the plasma membrane of Leishmania as revealed by fracture-flip.

We used a modification of fracture-flip to reveal the nanoanatomy of the inner surface of the plasma membrane in promastigotes of Leishmania. After freeze-fracture, lightly fixed promastigotes were coated with a stabilizing layer of carbon evaporated from an electron gun, thawed, and washed. Fractured promastigotes attached to the carbon casts by the protoplasmic (i.e., inner) halves of their plasma membranes were treated with Triton X-100, followed by exposure to low concentrations of trypsin and thorough washing. This was followed by picking up and flipping of the replicas, followed by air-drying. The actual inner surfaces of the plasma membrane were then imaged by platinum shadowing. Extended, three-dimensional, high-resolution views of the inner surface of the plasma membrane showed parallel arrays of microtubules (average spacing 47 nm) closely apposed to the inner surface. Cytochemical labeling confirmed the morphological identification of both subpellicular and flagellar microtubules, as determined by treatment with mouse monoclonal anti-alpha- or anti-beta-tubulin, followed by labeling with goat anti-mouse IgG adsorbed to colloidal gold. Removal of the microtubules revealed parallel arrays of particles (average diameter 17 nm). We hypothesize that these particles represent the cytoplasmic portion of proteins that link the microtubules to the plasma membrane.     

Double replica electroblotting: a method to produce two replicas from one gel

Electroblotting is a method by which proteins or nucleic acids, separated by electrophoresis, are transferred, also by electrophoresis, from a gel to a so-called transfer medium, e.g a nitrocellulose membrane. In some experiments, it is desirable to be able to obtain more than one replica from each gel and it has now proved possible to produce two replicas, which are almost identical, from one gel. This is achieved by applying one membrane on each side of the gel and change the direction of the current several times in such a way that the efficient transfer time is short in the beginning of the electroblotting and is increased for each cycle. This procedure will be referred to as 'double replica electroblotting'. Proteins were transferred at 100 V and the duration of an experiment with 2 h efficient transfer time in each direction was 7 h. The gel was more efficiently depleted of proteins after double replica electroblotting as compared to ordinary electrotransfer in one direction. Cathodically migrating proteins are also trapped on the membranes with this technique. Double replica electroblotting was used to produce two replicas from ordinary sodium dodecyl sulfate polyacrylamide gels as well as from 2-dimensional gels.     

Purified lac permease and cytochrome o oxidase are functional as monomers

Purified lac permease, the 46.5-kDa product of the lac Y gene that catalyzes lactose/H+ symport, or purified cytochrome o, a terminal oxidase of the Escherichia coli respiratory chain composed of four subunits with a composite molecular mass of 140 kDa, was reconstituted into proteoliposomes individually or in combination. The preparations were then examined by freeze-fracture electron microscopy employing conventional platinum/carbon replicas or by means of a new technique using thin tantalum replicas. In nonenergized proteoliposomes, both proteins appear to reconstitute as monomers based on (i) the variation of intramembrane particle density with protein concentration; (ii) the ratio of particles corresponding to each protein in proteoliposomes reconstituted with a known ratio of permease to oxidase; and (iii) the dimensions of the particles observed in tantalum replicas. The intramembrane particle diameters in tantalum replicas are about 20-25% smaller than those observed in conventional platinum/carbon replicas, indicating that the dimensions of the particles revealed with tantalum more accurately reflect the sizes of lac permease and cytochrome o. The diameters and heights of the permease and cytochrome o in tantalum replicas are 5.1 nm X 2.8 nm and 7.4 nm X 4.2 nm, respectively. Furthermore, a higher percentage of lac permease molecules exhibits a notch or cleft in tantalum replicas relative to platinum/carbon replicas. Importantly, the initial rate of lactose/H+ symport in proteoliposomes varies linearly with the ratio of lac permease to phospholipid, and no change is observed in either the size or distribution of lac permease molecules when the proteoliposomes are energized. The results taken as a whole provide a strong indication that both lac permease and cytochrome o reconstitute into proteoliposomes as monomers, that the permease does not dimerize in the presence of the H+ electrochemical gradient, and that both molecules are completely functional as monomers.     

Label-fracture of cell surfaces by replica staining

We introduce replica-staining label-fracture, a method for the cytochemical mapping of membrane surfaces. This method is a corollary of the rationale of label-fracture (Pinto da Silva and Kan, 1984: J Cell Biol 99:1156). After freeze-fracture the exoplasmic halves of the membrane remain attached to the replica. We show that cytochemical labeling of cell surfaces can be performed by direct post-fracture staining of freeze-fracture replicas. This new variant of label-fracture leads to miniaturization of labeling procedures and allows standardization of labeling conditions and simultaneous processing of different specimens.     

Preparing Mixed Cellulose Ester or Polycarbonate Membrane Filter Replicas for Transmission Electron Microscopy

Simple methods for preparing large numbers of grids exhibiting excellent coverage of intact replicas on mixed cellulose ester or polycarbonate membrane filters are described. The techniques ensure that grids and carbon replicas receive identical treatment and are not rearranged or lost during processing. The techniques permit grids and filter sections to be handled en masse rather than individually. Also, replica section squares remain centered on the grids. A temporary grid storage method (“grid- pad”) is also described, which facilitates grid identification and handling.     

A new method to visualize the Helicobacter pylori-associated Lewis(b)-binding adhesin utilizing SDS-digested freeze-fracture replica labeling.

Freeze-fracture replica labeling has become a versatile tool to visualize both membrane components and other cell structures using SDS-replica cleaning before specific immunogold labeling of proteins or lipids. We report here for the first time the adoption and optimization of the method to studies of bacterial envelopes, as applied to structural analysis of the distribution of the unique BabA-adhesin of the gastric pathogen Helicobacter pylori. BabA is important for bacterial adherence to the human epithelial cell lining of the stomach. The adhesin was found to be distributed all over the bacterial cell surfaces. Our results suggest that the SDS-replica labeling allows assessment of protein localization to distinct cell compartments and analysis of co-localization with neighboring membrane structures.     

Stable carbon isotope fractionations of the hyperthermophilic crenarchaeon Metallosphaera sedula

The stable carbon isotopic compositions of the inorganic carbon source, bulk cell material, and isoprenoid lipids of the hyperthermophilic crenarchaeon Metallosphaera sedula, which uses a 3-hydroxypropionate-like pathway for autotrophic carbon fixation, have been measured. Bulk cell material was approximately 3 per thousand enriched in 13C relative to the dissolved inorganic carbon, and 2 per thousand depleted in 13C relative to isoprenoid membrane lipids. The isotope data suggested that M. sedula uses mainly bicarbonate rather than CO(2) as inorganic carbon source, which is in accordance with a 3-hydroxypropionate-like carbon fixation pathway. To the best of our knowledge this is the first report of 13C fractionation effects of such a hyperthermophilic crenarchaeon.     

Freeze-fracture cytochemistry: a new fracture-labeling method for topological analysis of biomembrane molecules

Freeze-fracture cytochemistry allows visualization of cellular and molecular characteristics of biomembranes in situ. In this review, we discuss freeze-fracture cytochemistry with special reference to a new cytochemical labeling of replicas, the detergent-digestion fracture-labeling technique. In this procedure, unfixed cells are rapidly-frozen, freeze-fractured, and physically stabilized by evaporated platinum/carbon. The frozen cells are then removed from the freeze-fracture apparatus to thaw and are subsequently treated with detergents. After detergent-digestion, replicas are labeled with cytochemical markers. We demonstrate that the technique is a versatile tool for direct analysis of the macromolecular architecture of biomembranes and allows identification of particular intracellular membrane organelles. In addition, we demonstrate the application of ultrasmall gold to freeze-fracture immunocytochemistry. Freeze-fracture cytochemistry is a valuable technique for investigating topology and dynamics of membrane molecules.     

A new type of substratum adhesion structure in NRK cells revealed by correlated interference reflection and electron microscopy

The substratum adherent membrane of NRK cells in vitro has been studied using correlated interference reflection and surface replica electron microscopy. Structures visualized by interference reflection microscopy were identified in the subsequently prepared platinum-carbon replicas of the adherent membranes. Substratum adherent membranes were prepared using a hypotonic shock/cell shearing technique (lysis-squirting). Typically 20% of the original ventral membrane surface area and 50% of the original focal adhesion number were retained. Microfilament bundle termini, clathrin-coated sheets and pits, cytotic vesicles having a ridged surface and groups of membrane associated particles were well preserved in the replicas. Two types of isolated adhesion structures were found after lysis-squirting. In addition to typical elongate focal adhesions containing actin and vinculin, we report the existence of adherent membrane patches lacking microfilament bundles and negative for vinculin labelling, but coated with clathrin and identifiable in interference reflection microscopy as less dark than focal adhesions and having dot, U- or sinusoidal shapes.     

High-resolution quantitative visualization of glutamate and GABA receptors at central synapses

Glutamate and GABA are the main transmitters in the central nervous system and their effects are mediated by ionotropic and metabotropic receptors. Immunogold electron microscopy has revealed the quantitative localization of these receptors at 20-30nm resolution. SDS-digested freeze-fracture replica labeling (SDS-FRL), a newly developed immunogold method, provides an accurate estimate of molecule numbers. Here, we summarize the recent advances in quantitative receptor localization, including use of SDS-FRL analyses to determine numbers of AMPA-type glutamate receptors in the cerebellum. The two-dimensional view and high sensitivity of SDS-FRL have revealed small, irregularly shaped AMPA receptor clusters within cerebellar synapses.     

Lipid composition of the isolated rat intestinal microvillus membrane

1. Rat intestinal microvillus plasma membranes were prepared from previously isolated brush borders and the lipid composition was analysed. 2. The molar ratio of cholesterol to phospholipid was greatest in the membranes and closely resembled that reported for myelin. 3. Unesterified cholesterol was the major neutral lipid. However, 30% of the neutral lipid fraction was accounted for by glycerides and fatty acid. 4. Five phospholipid components were identified and measured, including phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, sphingomyelin and lysophosphatidylcholine. Though phosphatidylethanolamine was the chief phospholipid, no plasmalogen was detected. 5. In contrast with other plasma membranes in the rat, the polar lipids of the microvillus membrane were rich in glycolipid. The cholesterol:polar lipid (phospholipid+glycolipid) ratio was about 1:3 for the microvillus membrane. Published data suggest that this ratio resembles that of the liver plasma membrane more closely than myelin or the erythrocyte membrane. 6. The fatty acid composition of membrane lipids was altered markedly by a single feeding of safflower oil. Membrane polar lipids did not contain significantly more saturated fatty acids than cellular polar lipids. Differences in the proportion of some fatty acids in membrane and cellular glycerides were noted. These differences may reflect the presence of specific membrane glycerides.     

The rat retina is a useful in vivo model to study membrane lipid synthesis: Rates of biosynthesis of neutral glycerides and phospholipids

The phospholipid composition was studied in the whole rat retina, as well as in its subcellular fractions. A relative enrichment of phosphatidic acid, phosphatidylethanolamine, and phosphatidylserine was observed in rod outer segments (ROS) in comparison with entire retina: nuclear-photoreceptor inner segmentssynaptic bodies (P₁) and synaptosomal-mitochondrial (P₂) fractions. Phosphatidylcholine was the predominant phospholipid class found in all subcellular fractions analyzed. The microsomal fraction was relatively enriched in phosphatidic acid and in phosphatidylinositol. In addition, the rat eye has been used as an in vivo system to study membrane lipid synthesis. After intravitreal injections of [2-³H]glycerol a rapid labeling of retinal glycerolipids took place. Up to 120 min after injection only the glycerol backbone of lipids was labeled. Phosphatidic acid and diacylglycerol displayed rapid rates of synthesis and breakdown. Fastest rates of labeling were attained by phosphatidylcholine followed by phosphatidylinositol. Differences were found when in vitro labeling by [2-³H]glycerol was compared with intravitreal injections. Labeling of phospholipids of subcellular fractions by intravitreally injected [2-³H]glycerol showed that most of the label accumulated in microsomal phosphatidylcholine and phosphatidylinositol. Diacylglycerols and phosphatidylethanolamine also took up 10 and 20% respectively of the precursor. It is concluded that the rat eye is a useful experimental model to study synthesis and metabolism of membrane lipids in the retina.     

Aminophospholipids have no access to the luminal side of the biliary canaliculus: implications for thr specific lipid composition of the bile fluid

About 95% of the bile phospholipids are phosphatidylcholine. Although the fractions of phosphatidylcholine and of both aminophospholipids phosphatidylserine and phosphatidylethanolamine in the canalicular membrane are in the same order of about 35% of total lipids, both aminophospholipids are almost absent from the bile. To rationalize this observation, we studied the intracellular uptake of various fluorescent phospholipid analogues and their subsequent enrichment in the bile canaliculus (BC) of HepG2 cells. Diacylaminophospholipid analogues but not phosphatidylcholine analogues became rapidly internalized by an aminophospholipid translocase (APLT) activity in the plasma membrane of HepG2 cells. We observed only low labeling of BC by diacylaminophospholipids but extensive staining by phosphatidylcholine analogues. In the presence of suramin, known to inhibit APLT, a strong labeling of BC by diacylaminophospholipid analogues was found that declined to a level observed for control cells after removal of suramin. Unlike diacylphosphatidylserine, diether phosphatidylserine analogue, which is not an appropriate substrate of APLT, accumulated in the BC. The correlation between low labeling of BC and an APLT-mediated transbilayer movement suggests the presence of an APLT activity in the canalicular membrane that prevents exposure of aminophospholipids to the bile.     

Preparation of apical plasma membranes from cells grown on coverslips. Electron microscopic investigations of the protoplasmic surface

We introduce here a simple method which permits an efficient isolation of apical plasma membranes from tissue culture cells and the electron microscopic examination of their protoplasmic surfaces by use of the platinum/carbon replica technique. Different procedures were tested with regard to the efficiency of isolation and preservation of ultrastructure. Best results were obtained by prestabilization of cell surfaces with low concentrations of tannic acid prior to isolation. To demonstrate the possible applications and versatility of the method, studies were done on virus-infected cells in combination with immunocytochemical labeling. With this model system, we show that it is possible to correlate the structures seen on the cytoplasmic surface of the plasma membrane with the distribution of virus antigens at the cell surface labeled with immunogold markers prior to preparation.     

Influence of enzymatic phospholipid cleavage on the permeability of the erythrocyte membrane. II. Protein-mediated transfer of monosaccharides and anions

In order to investigate the influence of membrane lipids on transport via the protein domain of the erythrocyte membrane, a number of facilitated diffusion processes was studied by tracer flux techniques in whole cells after cleavage of up to 65% of the phosphatidylcholine or the sphingomyelin by phospholipase A₂ from Naja naja or bee venom, or by sphingomyelinase, respectively.The mediated fluxes of l-arabinose, which is transported by the glucose carrier, and of l-lactate, which uses a specific monocarboxylate carrier, were markedly inhibited by cleavage of either phosphatidylcholine or sphingomyelin. These phospholipid dependencies are in line with earlier data on cholesterol dependencies (Deuticke, B. (1977) Rev. Physiol. Biochem. Pharmacol. 78, 1–97). They can only in part be explained by changes of membrane fluidity. More specific interactions of the degradation products with the carrier proteins seem also to play a role.Sulfate and oxalate transfer, which proceed via the inorganic anion-exchange system, are essentially unaffected by cleavage of phosphatidylcholine and less sensitive to sphingomyelin cleavage than the two other processes. This also agrees with earlier data on cholesterol independency of sulfate transfer. The inorganic anion-exchange protein thus seems to be less dependent on the surrounding lipids in its conformation and its mode of action than the two other carriers.     

Molecular cytochemistry of CD3 and CD4 antigens in human lymphocytes as studied by label-fracture and by fracture-label

Label-fracture and fracture-label membrane immunocytochemistry are used to analyze the surface distribution, dynamics and partition on fracture of CD3 and CD4 antigens of human T lymphocytes. Redistribution of the antigens, induced by treatment at 37 degrees C with specific monoclonal antibodies, results in patching and capping of the labeling as observed in label-fractured specimens. Examination of platinum/carbon replicas of freeze-fractured plasma membranes of antibody-treated cells does not reveal recognizable domains of intramembrane particles. However, in cells where the aggregation of intramembrane particles is induced by incubation with glycerol, colloidal gold-labeled CD3 and CD4 molecules are seen confined to particulate domains of the membrane. Therefore, the lack of visible aggregation of intramembrane particles in patched or capped regions of the membrane implies that migration of CD3 and CD4 antigens with concentration in domains of the membrane is achieved contemporaneously with export of other non-capped integral membrane proteins from the same regions, in a process of diffusional equilibrium. Examination of fracture-labeled specimens shows that CD4 molecules partition on fracture with the inner protoplasmic face of the plasma membrane. This partition illustrates the transmembrane attitude of the antigen molecule and is a probable consequence of interaction of the protein with other components of the membrane or with the cytoskeleton.     

Membrane-rigidifying effects of anti-cancer dietary factors

Since several anti-cancer drugs interact with cell membrane lipids, the effects of anti-cancer dietary factors on liposomal membranes with different lipid composition were comparatively studied by measuring fluorescence polarization. Fluidity was imparted on both hydrophobic and hydrophilic regions of lipid bilayers by decreasing cholesterol and increasing unsaturated phosphatidylcholine in membranes. At 0.625-10 microM, (-)-epigallocatechin gallate, genistein, apigenin, resveratrol and a reference anti-cancer drug, doxorubicin, rigidified the tumor cell model membranes consisting of 20 mol% cholesterol and 80 mol% phosphatidylcholine with the acyl chain 18:1/16:0 ratio of 1.0, but not daidzein. They were more effective on the membrane core than the membrane surface. Quercetin showed a biphasic effect on the hydrophobic regions of membrane lipid bilayers to rigidify above 5 microM and fluidize below 2.5 microM. In contrast, anti-cancer dietary factors and doxorubicin were not or much less effective in rigidifying the normal cell model membranes consisting of 40 mol% cholesterol and 60 mol% phosphatidylcholine with the acyl chain 18:1/16:0 ratio of 0.5. The membrane-rigidifying effects were greater depending on a decrease of the cholesterol/phosphatidylcholine ratio and an increase of the phosphatidylcholine unsaturation degree. Membrane-active dietary factors and doxorubicin inhibited the growth of mouse myeloma cells at 10-100 microM, while the growth inhibition by membrane-inactive daidzein was relatively weak. Anti-cancer dietary factors appear to act on more fluid membranes like tumor cells as well as doxorubicin to induce rigidification, especially in the hydrocarbon core of membrane lipids, which is determined by the composition of cholesterol and unsaturated phospholipids.