Freeze-etch Electron Microscopy Research Articles

Osmotic behavior of erythrocytes from patients with a major affective disorder: A freeze-fracture electron microscopic study

Previously we have demonstrated a state-dependent decrease in the number of membrane vesicles in erythrocytes from patients with a major depressive episode. We now report an increase in the number of membrane vesicles during a manic episode as well as a reduction during lithium treatment and we also present data suggesting that the number of erythrocyte membrane vesicles in the affective disorders is dependent on osmotic shrinkage due to the freezing for freeze-etch electron microscopy. Although caution is required since the interrater reliability of the measurement of osmotic strain was insufficient in the mid range where it was tested, we do not think this invalidates the differences in osmotic strain found in the low and high ranges during depressive and manic episodes respectively. These findings warrant the use of more precise techniques in studies of the osmotic behavior of erythrocytes from patients with a major affective disorder.

Three-Dimensional Organization of Extracellular Matrix in Elastic Cartilage as Viewed by Quick Freeze, Deep Etch Electron Microscopy

Freeze-etch electron microscopy was used to reexamine the ultrastructure of extracellular matrix in elastic cartilage. This revealed that extremely delicate, approximately 4 nm diameter fibrils join end-to-end and sometimes side-to-side to form a tightly woven mesh that extends continuously from the cell membrane throughout the intercellular space. Within this meshwork were found large, irregularly contoured and densely packed elastin fibers as well as long, thin (20 nm) fibers with the appearance of type II collagen. By comparison, type I collagen fibers found in the skin surrounding the cartilage appeared much thicker (30 nm) and displayed the usual periodic banding pattern. Freeze-etching the latter fibers displayed a helicoidal arrangement of subfibrils within. In both cartilage and type I collagen-rich extracellular matrix, some of the approximately 4 nm filaments in the matrix could be seen to contact collagen fibers orthogonally, apparently connecting adjacent fibers at regular intervals. The organization of these fine filaments and others composing the matrix has several features that suggest a different organization for cartilage than currently thought. Specifically, the distance between branch or contact points of fibrils in the matrix is seldom more than 35 nm, substantially less than the length of one extended proteoglycan monomer. This suggests that other proteoglycans, or other unidentified components of the matrix, bind along the proteoglycan core protein at intermediate binding sites in order to form a finely partitioned structure.

Visualization of RecA protein and its complexes with DNA by quick-freeze/deep-etch electron microscopy

Freeze-etch electron microscopy of pure RecA protein aggregates, as well as of RecA protein complexes on single-stranded and double-stranded DNA formed with various nucleotides, has permitted a clearer discrimination between the two different helical polymers that this protein forms. Both are continuous, single-start, right-handed helices; however, the form observed when ATP or non-hydrolyzable ATP analogs are present has a pitch of 9.5 nm and a diameter of 10 nm, while the other form, observed in the absence of ATP or its analogs, or in the presence of ADP, has a pitch of 6 nm and a diameter of 12 nm. The former “long pitch” helix is found only when RecA protein is bound to DNA. The latter “short pitch” helix is also observed in pure RecA protein polymers (also termed rods) and in the needlelike paracrystals of RecA protein that form in the presence of magnesium or spermidine ions, representing bundles of rods closely packed in register. Addition of ATP or nonhydrolyzable ATP analogs in the absence of DNA dissociates the pure RecA protein crystals, as well as individual helical rods, into short curvilinear chains of attached monomers. These chains typically form closed, circular rings of 7(±1) protein monomers, similar in construction to a single turn of the RecA protein helix, but significantly broader in diameter. The role of ATP in interconverting the various polymeric forms of RecA protein is discussed within the context that ATP functions as a reversible allosteric effector of RecA protein, much as it mediates reversible conformational changes in other vectoral motor proteins such as myosin, dynein, kinesin and the 70,000 Mr “heat shock” ATPases. We discuss how cyclic conversions back and forth between the short- and long-pitch conformations of RecA protein could mediate in reversible single-stranded and double-stranded DNA interactions during the search for homology.

Localization and characterization of intracellular liquid-liquid phase separations in deeply frozen populus using electron microscopy, dynamic mechanical analysis and differential scanning calorimetry

Populus balsamifera var. virginiana (Sargent), balsam poplar, is capable of withstanding liquid nitrogen temperatures when cooling is slow (less than 6°C h −1) and the twigs are winter dormant. Cooling to −50°C at rates as high as 42°C h −1 is also tolerated. The mechanical behavior during warming of dormant twigs cooled at rates varying from 3°C h −1 to 600 °C h −1 has been investigated using resonance mode dynamic mechanical analysis (DMA). The results were correlated with those of freeze-etch electron microscopy investigations of the samples as well as differential scanning calorimetry (DSC) of model solutions. The results of these investigations were compared with mortality data on the twigs. This has led us to hypothesize that at critical cooling rates below 0° C plasma membranes tear, leading to the rapid growth of intracellular ice. This in turn prevents the intracellular liquid-liquid phase separations seen by DMA in samples cooled at sublethal rates. DSC analysis of salt-sugar-protein model solutions supports the idea that when liquid-liquid phase separation does occur, some domains are sugar-depleted but rich in protein, while others are sugar-rich but low in protein. This may have important implications regarding the organization of extreme freezing resistance mechanisms in this natural system.

Trimeric binding of the 70-kD uncoating ATPase to the vertices of clathrin triskelia: a candidate intermediate in the vesicle uncoating reaction.

Clathrin-coated vesicles were uncoated with the 70-kD "uncoating ATPase" from bovine brain, and the molecular products were visualized by freeze-etch electron microscopy. This yielded images of released clathrin triskelia with up to three 70-kD uncoating ATPase molecules bound to their vertices. Likewise, incubation of soluble clathrin triskelia with purified uncoating ATPase also led to trimeric binding of the ATPase to the vertices of clathrin triskelia. However, this occurred only when either EDTA or nonhydrolyzable analogues of ATP were present, in which case the ATPase also appeared to self-associate. When ATP was present instead, no 70-kD ATPases could be found on clathrin triskelia and all ATPases remained monomeric. These observations support the notion that ATP controls an allosteric conversion of the 70-kD uncoating ATPase between two different molecular conformations, an ATP-charged state in which the molecule has relatively low affinity for itself as well as low affinity for clathrin, and an ATP-discharged state in which both of these affinities are high. We presume that in vivo, the latter condition is brought about by ATP hydrolysis and product release, at which point the ATPase will bind tightly to clathrin and/or self-associate. We further propose that these reactions, when occurring in concert within a clathrin lattice, will tend to destabilize it by a mechanism we call "protein polymer competition". We stress the analogies between such a mechanism of uncoating and the ATP-driven events in muscle contraction. Finally, we show that under experimental conditions in which the uncoating ATPase fully removes the coats from brain coated vesicles, identical aliquots of the enzyme do not affect plasmalemmal coated pits in situ. This remarkable selectivity, the mechanism of which remains a complete mystery, is at least consistent with the idea that the 70-kD ATPase indeed plays a role in uncoating coated vesicles after they have formed in vivo.

Involvement of plasma membrane complexes in cellulose deposition in desmids

Deposition of cellulose microfibrils in oriented patterns in plant cell walls is believed to be one of the mechanisms by which cell shape is controlled. In some desmids, such as Closterium. circumferentially oriented microfibrils appear to help generate an elongated spindle shaped cell. In contrast, the primary cell wall of Micrasterias contains an apparently random meshwork of microfibrils, yet an elaborate multi-lobed semicell is formed. Freeze-etch electron microscopy of unfixed, ultrarapidly frozen desmids was undertaken to identify plasma membrane and cytoskeletal elements that may participate in the deposition and orientation of cellulose microfibrils.Micrasterias denticulata and aClosterium sp. were grown under a light/dark cycle to induce partial synchronization of cell division. Groups of several cells at the desired stage of semicell regeneration were loaded between two modified Balzers gold planchets coated with a slurry of yeast. A slot grid spacer prevented the cells from being crushed. Specimens were frozen in a propane jet freezing device designed by Gilkey and Staehelin. Replicas were produced in a Balzers BA 360 freeze-etch device and examined in a JEOL 100 CX electron microscope.

Physical biochemistry of a liposomal amphotericin B mixture used for patient treatment

There seems little doubt now that intravenous liposomal amphotericin B can be a useful treatment modality for the management of immunocompromised patients with suspected or proven disseminated fungal infections. Interestingly, the very significant reduction in toxicity reported when amphotericin B is part of a bilayer membrane is closely tied to the physical characteristics of the liposomes involved, although these are poorly understood at the molecular level. We record here an examination by spectroscopy and freeze-etch electron microscopy of unsonicated amphotericin B multilamellar vesicles prepared along the lines that we and others have followed for samples used in clinical trials and preclinical in vivo or in vitro studies. Our study has focussed on liposomes of 7:3 dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DMPC/DMPG) bearing 0-25 mol% amphotericin B, since this lipid mixture has been the choice for the first clinical trials. Phase transition behaviour of these liposomes was examined by electron paramagnetic resonance (EPR) spectroscopy of a nitroxide spin label partitioning into the bilayers. The same experiments were then performed on similarly prepared liposomes of the disaturated species, dipalmitoylphosphatidylcholine (DPPC), and the diunsaturated species, dielaidoylphosphatidylcholine (DEPC). Partial phase diagrams were constructed for each of the lipid/drug mixtures. Melting curves and derived phase diagrams showed evidence that amphotericin B is relatively immiscible with the solid phase of bilayer membranes. The phase diagram for DEPC/amphotericin B was very similar to that of DPPC/amphotericin B, and both exhibited less extensive temperature ranges of phase separation than did the 7:3 DMPC/DMPG mixture with amphotericin B. Between 25 and 37 degrees C the measured fluidity of the 7:3 DMPC/DMPG liposomes was similar to that of the (unsaturated fatty acid) DEPC liposomes, and considerably higher than that seen for (saturated fatty acid) DPPC liposomes. Preparations of 7:3 DMPC/DMPG, DPPC, and DEPC containing 0-25 mol% amphotericin B were examined by freeze-etch electron microscopy at 35 and 22 degrees C (to cover the temperature range of the mammalian body core and periphery). The same liposome features were present in all three liposome types studied. The appearance of individual liposomes at x 100,000 magnification reflected their molecular characteristics, which were found to be significantly heterogeneous within each batch. The lipid/drug structures were bilayer in nature, although liposomes showing considerable disruption were common, particularly at the highest drug concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)

A liposomal MRI contrast agent: phosphatidylethanolamine-DTPA.

The chelating agent, diethylenetriaminepentaacetic acid (DTPA), was attached via one -COOH group to the amino headgroup of phosphatidylethanolamine to produce a phospholipid which is also a powerful chelating agent. It readily assembles into the walls of lipid bilayer structures as a liposome-associated carrier of cations for MR contrast or radioisotope studies. Freeze-etch electron microscopy showed that phosphatidylethanolamine-DTPA formed satisfactory sonicated vesicles when mixed with natural phospholipids at up to 50 wt%. The resultant structures with bound gadolinium effectively shortened T1 and T2 of surrounding water protons. When sonicated liposomes bearing chelating agent with bound 111In3+ were injected intravenously into rats, uptake was primarily by liver and spleen. By 24 h postinjection there was biliary excretion of this material. Phosphatidylethanolamine-DTPA may have some general utility as an amphiphilic liposomal chelating agent for polyvalent cations.

Persistence at low temperature of the P β′ ripple in dipalmitoylphosphatidylcholine multilamellar vesicles containing either glycosphingolipids or cholesterol

The disappearance and reappearance of the P β′ ripple in multilamellar liposomes of dipalmitoylphosphatidylcholine (DPPC) has been examined by freeze-etch electron microscopy. The presence of less than 10 mol% of various glycosphingolipids or cholesterol in the liposomes markedly increases the time required for ripple disappearance when the vesicles are cooled from 38°C to 30°C, as compared to the pure phospholipid. Once the ripples have begun to disappear in the two-component vesicles, they do not uniformly reappear until the system is heated above the main transition of DPPC and allowed to cool into the pertransition region. These results suggest that the long time for ripple disappearance in the two-component systems reflects a slow molecular reorganization process which occurs when the systems are forced to change from the P β′ gel to the L β′ gel by a temperature downshift.

Hypertonic media inhibit receptor-mediated endocytosis by blocking clathrin-coated pit formation.

Two seemingly unrelated experimental treatments inhibit receptor mediated endocytosis: (a) depletion of intracellular K+ (Larkin, J. M., M. S. Brown, J. L. Goldstein, and R. G. W. Anderson. 1983. Cell. 33:273-285); and (b) treatment with hypertonic media (Daukas, G., and S. H. Zigmond. 1985. J. Cell Biol. 101:1673-1679). Since the former inhibits the formation of clathrin-coated pits (Larkin, J. M., W. D. Donzell, and R. G. W. Anderson, 1986. J. Cell Biol. 103:2619-2627), we were interested in determining whether hypertonic treatment has the same effect, and if so, why. Fibroblasts (human or chicken) were incubated in normal saline made hypertonic with 0.45 M sucrose, then broken open by sonication and freeze-etched to generate replicas of their inner membrane surfaces. Whereas untreated cells display typical geodesic lattices of clathrin under each coated pit, hypertonic cells display in addition a number of empty clathrin "microcages". At first, these appear around the edges of normal coated pit lattices. With further time in hypertonic medium, however, normal lattices largely disappear and are replaced by accumulations of microcages. Concomitantly, low density lipoprotein (LDL) receptors lose their normal clustered distribution and become dispersed all over the cell surface, as seen by fluorescence microscopy and freeze-etch electron microscopy of LDL attached to the cell surface. Upon return to normal medium at 37 degrees C, these changes promptly reverse. Within 2 min, small clusters of LDL reappear on the surfaces of cells and normal clathrin lattices begin to reappear inside; the size and number of these receptor/clathrin complexes returns to normal over the next 10 min. Thus, in spite of their seeming unrelatedness, both K+ depletion and hypertonic treatment cause coated pits to disappear, and both induce abnormal clathrin polymerization into empty microcages. This suggests that in both cases, an abnormal formation of microcages inhibits endocytosis by rendering clathrin unavailable for assembly into normal coated pits.

Assembly of complement components C5b-8 and C5b-9 on lipid bilayer membranes: visualization by freeze-etch electron microscopy.

We have visualized by freeze-etch electron microscopy the macromolecular complexes of complement, C5b-8 and C5b-9, respectively, assembled on synthetic phospholipid bilayers. These complexes were formed sequentially by using purified human complement components C5b-6 followed by C7, C8, and C9. Complexes of C5b-8 were observed on the external surface (ES) of vesicles as 12-nm particles that tended to form polydisperse aggregates. The aggregates were sometimes of a regular chainlike structure containing varying numbers of paired subunits. Etching of vesicles containing C5b-9 complexes revealed on the ES large rings of approximately 27-nm outer diameter. One or two knobs usually were attached to the perimeter of the rings. Splitting of the membrane resulted in partitioning of the C5b-9 with the outer leaflet. Thus, round holes of approximately 17-nm diameter were present in the protoplasmic face (PF), and raised circular stumps of a matching size were present on the exoplasmic face (EF) of C5b-9 vesicles. C5b-9 complexes were frequently localized in regions of the lowest lipid order. That is, in micrographs of the EF and ES, single C5b-9 complexes were located where the ripples of the P beta' phase bend or reach a dead end, and linear arrays of C5b-9 complexes outlined disclination-like structures in the lattice; the holes in the PF mirrored this distribution. The membrane immediately surrounding C5b-9 rings was often sunk inwardly over an area much larger than that of the ring itself.(ABSTRACT TRUNCATED AT 250 WORDS)

MULTIPLE EMULSIONS STABILIZED BY COLLOIDAL MICROGRYSTALLINE CELLULOSE

ABSTRACT Multiple emulsions stabilized by colloidal microcrystalline cellulose (CMCC, Avicel RC591) at the w/o and o/w interfaces, and by the addition of Span 80 or Span 85 at the w/o interface, were studied by means of brightfield microscopy, freeze-etch electron microscopy, droplet size distribution analysis and rheologic measurements. Stable multiple emulsions were prepared by incorporation of sodium chloride in the innermost aqueous phase, thereby creating an osmotic gradient preventing loss of the inner aqueous phase to the external aqueous phase. Freeze-etch electron microscopy of the multiple emulsions indicated the presence of a network of microcrystalline cellulose at the outer o/w interface. It may be assumed that the surfactant directly stabilized the w/o interface by adsorption at the interface, as well as indirectly by facilitating wetting of the microcrystalline cellulose by the oil. From rheologic measurements, the existence of a three-dimensional network in the external aqueous phase was indicated by the considerable degrees of thlxotropy and significant static yield values of these multiple emulsions.

Deep-etch visualization of proteins involved in clathrin assembly.

Assembly proteins were extracted from bovine brain clathrin-coated vesicles with 0.5 M Tris and purified by clathrin-Sepharose affinity chromatography, then adsorbed to mica and examined by freeze-etch electron microscopy. The fraction possessing maximal ability to promote clathrin polymerization, termed AP-2, was found to be a tripartite structure composed of a relatively large central mass flanked by two smaller mirror-symmetric appendages. Elastase treatment quantitatively removed the appendages and clipped 35 kD from the molecule's major approximately 105-kD polypeptides, indicating that the appendages are made from portions of these polypeptides. The remaining central masses no longer promote clathrin polymerization, suggesting that the appendages are somehow involved in the clathrin assembly reaction. The central masses are themselves relatively compact and brick-shaped, and are sufficiently large to contain two copies of the molecule's other major polypeptides (16- and 50-kD), as well as two copies of the approximately 70-kD protease-resistant portions of the major approximately 105-kD polypeptides. Thus the native molecule seems to be a dimeric, bilaterally symmetrical entity. Direct visualization of AP-2 binding to clathrin was accomplished by preparing mixtures of the two molecules in buffers that marginally inhibit AP-2 aggregation and cage assembly. This revealed numerous examples of AP-2 molecules binding to the so-called terminal domains of clathrin triskelions, consistent with earlier electron microscopic evidence that in fully assembled cages, the AP's attach centrally to inwardly-directed terminal domains of the clathrin molecule. This would place AP-2s between the clathrin coat and the enclosed membrane in whole coated vesicles. AP-2s linked to the membrane were also visualized by enzymatically removing the clathrin from brain coated vesicles, using purified 70 kD, uncoating ATPase plus ATP. This revealed several brick-shaped molecules attached to the vesicle membrane by short stalks. The exact stoichiometry of APs to clathrin in such vesicles, before and after uncoating, remains to be determined.

Unusual lipid structures selectively reduce the toxicity of amphotericin B.

Ribbon-like structures result when amphotericin B interacts with lipid in an aqueous environment. At high ratios of amphotericin to lipid these structures, which are lipid-stabilized amphotericin aggregates, become prevalent resulting in a dramatic attenuation of amphotericin-mediated mammalian cell, but not fungal cell, toxicity. Studies utilizing freeze-etch electron microscopy, differential scanning calorimetry, 31P NMR, x-ray diffraction, and optical spectroscopy revealed that this toxicity attenuation is related to the macromolecular structure of the complexes in a definable fashion. It is likely that amphotericin in this specific form will have a much improved therapeutic utility.

X-ray and dissolution studies of paramylon storage granules fromEuglena

Paramylon is the β-1,3 glucan storage carbohydrate in the euglenoid algae. Mature paramylon granules are highly crystalline, fibrillar, and have a complex substructure. X-ray diffraction was used to demonstrate that mature paramylon granules are much more crystalline than immature granules. Freeze-etch electron microscopy showed that in mature granules, the microfibrils are organized in highly ordered arrays while the microfibrils of immature granules are less organized. The data suggest that the high crystallinity of paramylon is due to higher-order aggregates of microfibrils and the interaction of water with the microfibrils. The dissolution of paramylon was recorded by darkfield videomicroscopy. In a 0.5 N NaOH solution, paramylon dissociates in a regular manner into its constituent 4 nm microfibrils, and the central region of the granule is the last remaining refractile area during the dissolution process.

Glycoprotein Filament Removal From Human Milk Fat Globules by Heat Treatment

Freeze-etch electron microscopy was applied to milk fat globules to observe surface details. A remarkable array of filaments, approximately 0.5 micron in length, was seen on human, but not bovine, globules. Heating human globules removed the filaments that were identified as high molecular weight glycoproteins by freeze-etch and gel electrophoretic analysis of the heating medium. Extraction of these globule glycoproteins was slight at 60 degrees C for one minute but substantial and tending to plateau at 80 degrees C for one minute. Such heat-induced alterations of the milk fat globule surface provide an alternative or additional explanation to milk lipase inactivation as the cause of reduced milk fat absorption from heated milk by the preterm infant.

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.

Structural localization of the O2-evolving apparatus to multimeric (tetrameric) particles on the lumenal surface of freeze-etched photosynthetic membranes.

Isolated appressed chloroplast membranes, highly enriched in photosystem II (PSII) activity, were examined by freeze-etch electron microscopy. The exposed surfaces of these Triton X-100 solubilized membrane fragments correspond to the lumenal or ESs surface of intact stacked thylakoid membrane regions (Dunahay, T. G., L. A. Staehelin, M. Seibert, P. D. Ogilvie, and S. P. Berg. 1984. Biochim. Biophys. Acta. 764:179-193). The sequential removal from this sample of three extrinsic proteins (17, 23, and 33 kD) associated with the O2-evolving apparatus and the concomitant loss of O2 evolution, was related to subtle changes in the height and substructure of characteristic multimeric (often tetrameric) particles that protrude from the ESs membrane surface. After removal of these proteins, the multimeric particles disappeared and dimeric particles of similar diameter but of lesser height (6.1 vs. 8.2 nm in the controls) were observed. Reconstitution of the depleted membrane fragments with the extrinsic proteins led to rebinding of the three proteins, to a 63% recovery of the control rates of O2 evolution, and to the reappearance of the larger multimeric particles. Analysis of the structural changes associated with the loss and rebinding of the extrinsic proteins is consistent with a stoichiometry of one PSII complex for either one or two copies of the 17-, 23-, and 33-kD proteins, and these are symmetrically arranged on the lumenal surface of the complex. These results demonstrate that the multimeric ESs particles correspond to part of the intact O2-evolving apparatus of PSII, thus confirming previous indirect studies relating these particles to PSII. The dimeric particles probably contain the rest of the O2-evolving complex.

Visualization of the cytoplasmic surface of Torpedo postsynaptic membranes by freeze-etch and immunoelectron microscopy.

The synapse-specific Mr 43,000 protein (43K protein) and the acetylcholine receptor were visualized by freeze-etch immunoelectron microscopy in preparations of purified Torpedo postsynaptic membranes. Vesicles were immobilized on glass and then sheared open by sonication to expose the cytoplasmic surface. Membranes were labeled with monoclonal antibodies to the 43K protein or the acetylcholine receptor. The cytoplasmic surface was devoid of filamentous structure, and the 43K protein and the cytoplasmic projection of the acetylcholine receptor were associated with prominent surface particles. Acetylcholine receptor and 43K protein, in membrane surfaces in direct contact with glass coated with polyornithine, segregated into dense particle aggregates separated by smooth membrane patches, whereas those in contact with glass coated with Alcian Blue underwent little or no detectable rearrangement. After treatment of vesicles at alkaline pH to remove the 43K protein, the cytoplasmic surfaces were still covered by a dense array of particles that were more uniform in shape and appeared slightly shorter than those seen on unextracted membranes, but similar in height to the extracellular projection. Monoclonal antibodies to the acetylcholine receptor labeled these particles, while antibodies to 43K protein did not. We conclude that the 43K protein is in direct association with the receptor and that complexes of the receptor and 43K protein can undergo surface-induced lateral redistribution. In addition, the cytoplasmic projection of the acetylcholine receptor is sufficiently large to be readily detected by freeze-etch electron microscopy and is similar in height to the extracellular projection.

Detection of protein-free lipoprotein analogues with an apolar lipid core by freeze-etch electron microscopy

Freeze-fracture electron microscopy of protein-free lipoprotein analogues consisting of polar phospholipids with neutral lipid cores shows that these systems can exhibit novel, smooth crossfracture faces. Resolution of these features can be enhanced by etching techniques. This novel particle morphology provides a new procedure to characterize microemulsions composed of polar and neutral lipids.