Freeze-fracture electron microscopy

The freeze-fracture technique consists of physically breaking apart (fracturing) a frozen biological sample; structural detail exposed by the fracture plane is then visualized by vacuum-deposition of platinum-carbon to make a replica for examination in the transmission electron microscope. The four key steps in making a freeze-fracture replica are (i) rapid freezing, (ii) fracturing, (iii) replication and (iv) replica cleaning. In routine protocols, a pretreatment step is carried out before freezing, typically comprising fixation in glutaraldehyde followed by cryoprotection with glycerol. An optional etching step, involving vacuum sublimation of ice, may be carried out after fracturing. Freeze fracture is unique among electron microscopic techniques in providing planar views of the internal organization of membranes. Deep etching of ultrarapidly frozen samples permits visualization of the surface structure of cells and their components. Images provided by freeze fracture and related techniques have profoundly shaped our understanding of the functional morphology of the cell.     

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     

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.     

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.     

GFP-tagged proteins visualized by freeze-fracture immuno-electron microscopy: a new tool in cellular and molecular medicine

GFP-tagging is widely used as a molecular tool to localize and visualize the trafficking of proteins in cells but interpretation is frequently limited by the low resolution afforded by fluorescence light microscopy. Although complementary thin-section immunogold electron microscopic techniques go some way in aiding interpretation, major limitations, such as relatively poor structural preservation of membrane systems, low labelling efficiency and the two-dimensional nature of the images, remain. Here we demonstrate that the electron microscopic technique freeze-fracture replica immunogold labelling overcomes these disadvantages and can be used to define, at high resolution, the precise location of GFP-tagged proteins in specific membrane systems and organelles of the cell. Moreover, this technique provides information on the location of the protein within the phospholipid bilayer, potentially providing insight into mis-orientation of tagged proteins compared to their untagged counterparts. Complementary application of the freeze-fracture replica immunogold labelling technique alongside conventional fluorescence microscopy is seen as a novel and valuable approach to verification, clarification and extension of the data obtained using fluorescent-tagged proteins. The application of this approach is illustrated by new findings on PAT-family proteins tagged with GFP transfected into fibroblasts from patients with Niemann-Pick type C disease.     

Quantitative retention of membrane lipids in the freeze-fracture replica

SDS-digested freeze-fracture replicas have been used as a substrate for immunoelectron microscopy to determine localization of membrane proteins and lipids. We, as well as others, have noticed that replicas prepared by first evaporating carbon are labeled more efficiently than conventional preparations in which platinum/carbon is evaporated first followed by carbon. In the present study, we examined whether the superior labeling in the carbon-first replica is caused by better retention of membrane molecules during SDS digestion. We used phosphatidylcholine liposomes as a model sample and measured the amount of inorganic phosphorus retained in the SDS-digested replica. The result showed that there was equivalent retention of inorganic phosphate among replicas prepared in different ways, indicating that labeling intensity on the replica did not correlate with the retention ratio. Interestingly, despite a similar retention ratio, replicas made of carbon alone gave far less labeling for ganglioside GM1 and phosphatidylcholine than replicas prepared by carbon followed by platinum/carbon. These results suggest that probes can bind to lipids captured by carbon more efficiently than those captured by platinum. Nonetheless, evaporation of platinum after carbon is indispensable for proper labeling.     

ELECTRON MICROSCOPE REPLICAS FROM THE SURFACE OF A FRACTURE THROUGH FROZEN CELLS

A technique is described, which is new in certain details, for fracturing frozen cells and taking replicas from the fracture surface. This technique has been developed as a cytological preparative technique complementary to those currently used in electron microscopy. The present paper describes results for human red cells, chosen for preliminary work to facilitate recognition of artifacts. The cells are suspended in 20 per cent glycerol, 0.9 per cent NaCl solution, allowed time to equilibrate, and rapidly frozen in a small chamber plunged into propane at -196°C. The chamber is kept at -196°C., and fractured in vacuo, in such a way that the fracture plane runs through the frozen suspension, and through the cells in its path. After fracture, the chamber is brought to -105°C., still in vacuo, to etch the fracture surface, and a platinum-carbon replica is deposited on the surface from a carbon arc. The replica is subsequently cleaned in concentrated alkaline solution and examined in the electron microscope. The outlines of the fractured cells can be recognised. There are indications that in areas of the replica which correspond to the interior of a cell, individual haemoglobin molecules can be seen, and in favourable cases the arrangement of some of the four molecular subunits.     

Growth substrate dependent localization of tetrachloroethene reductive dehalogenase in Sulfurospirillum multivorans

Sulfurospirillum multivorans is a dehalorespiring organism, which is able to utilize tetrachloroethene as terminal electron acceptor in an anaerobic respiratory chain. The localization of the tetrachloroethene reductive dehalogenase in dependence on different growth substrates was studied using the freeze-fracture replica immunogold labeling technique. When the cells were grown with pyruvate plus fumarate, a major part of the enzyme was either localized in the cytoplasm or membrane associated facing the cytoplasm. In cells grown on pyruvate or formate as electron donors and tetrachloroethene as electron acceptor, most of the enzyme was detected at the periplasmic side of the cytoplasmic membrane. These results were confirmed by immunoblots of the enzyme with and without the twin arginine leader peptide. Trichloroethene exhibited the same effect on the enzyme localization as tetrachloroethene. The data indicated that the localization of the enzyme was dependent on the electron acceptor utilized.     

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.     

Connexin Composition in Apposed Gap Junction Hemiplaques Revealed by Matched Double-Replica Freeze-Fracture Replica Immunogold Labeling

Despite the combination of light-microscopic immunocytochemistry, histochemical mRNA detection techniques and protein reporter systems, progress in identifying the protein composition of neuronal versus glial gap junctions, determination of the differential localization of their constituent connexin proteins in two apposing membranes and understanding human neurological diseases caused by connexin mutations has been problematic due to ambiguities introduced in the cellular and subcellular assignment of connexins. Misassignments occurred primarily because membranes and their constituent proteins are below the limit of resolution of light microscopic imaging techniques. Currently, only serial thin-section transmission electron microscopy and freeze-fracture replica immunogold labeling have sufficient resolution to assign connexin proteins to either or both sides of gap junction plaques. However, freeze-fracture replica immunogold labeling has been limited because conventional freeze fracturing allows retrieval of only one of the two membrane fracture faces within a gap junction, making it difficult to identify connexin coupling partners in hemiplaques removed by fracturing. We now summarize progress in ascertaining the connexin composition of two coupled hemiplaques using matched double-replicas that are labeled simultaneously for multiple connexins. This approach allows unambiguous identification of connexins and determination of the membrane "sidedness" and the identities of connexin coupling partners in homotypic and heterotypic gap junctions of vertebrate neurons.     

Fundamental Technical Elements of Freeze-fracture/Freeze-etch in Biological Electron Microscopy

Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated to generate a carbon/platinum “cast” intended for examination by transmission electron microscopy. Specimens are subjected to ultrarapid freezing rates, often in the presence of cryoprotective agents to limit ice crystal formation, with subsequent fracturing of the specimen at liquid nitrogen cooled temperatures under high vacuum. The resultant fractured surface is replicated and stabilized by evaporation of carbon and platinum from an angle that confers surface three-dimensional detail to the cast. This technique has proved particularly enlightening for the investigation of cell membranes and their specializations and has contributed considerably to the understanding of cellular form to related cell function. In this report, we survey the instrument requirements and technical protocol for performing freeze-fracture, the associated nomenclature and characteristics of fracture planes, variations on the conventional procedure, and criteria for interpretation of freeze-fracture images. This technique has been widely used for ultrastructural investigation in many areas of cell biology and holds promise as an emerging imaging technique for molecular, nanotechnology, and materials science studies.     

Freeze-fracture cytochemistry: a new method combining immunocytochemistry and enzyme cytochemistry on replicas.

We describe a new freeze-fracture cytochemical technique consisting of combined immunocytochemistry and enzyme cytochemistry. This technique reveals the relationship between molecules in biological membranes by double labeling with two different cytochemical markers (i.e., immunogold probes and cerium). In this method, antigens were detected with specific primary antibodies and appropriate secondary immunoprobes. Subsequently, alkaline phosphates activity was detected with cerium as the capture agent on the same replicas. Octyl-glucoside (OG) digestion before the cytochemical reactions was crucial to the success of this combined method. OG is an efficient detergent and OG digestion can preserve both immunocytochemical antigenicity and enzyme activity on replicas. As an initial examination, we applied this technique to the study of glycosyl-phosphatidyl-inositol-anchored proteins and adhesion molecules in human neutrophils. The method described here should serve as a unique additional approach for the study of topology and dynamics of molecules in biomembranes.     

High-resolution en-face visualization of the cardiomyocyte plasma membrane reveals distinctive distributions of spectrin and dystrophin

The actin-binding proteins, spectrin and dystrophin, are key components of the plasma membrane-associated cytoskeleton of the cardiac muscle cell. From confocal immunofluorescence studies, the distribution of spectrin appears to overlap with that of dystrophin, but the precise functional differentiation, molecular distributions and spatial relationship of these two cytoskeletal systems remain unclear. Freeze-fracture replica immuno-electron microscopy, in parallel with immunofluorescence/confocal microscopy, were applied to examine at high resolution the spatial relationships between the spectrin and dystrophin membrane-associated cytoskeleton systems in cardiac muscle. Application of freeze-fracture replica cytochemistry, with single and double immunogold labeling, permitted simultaneous examination of the organization of spectrin and dystrophin in en-face views of the plasma membrane at high resolution. In contrast to the close spatial relationship previously demonstrated for dystrophin and β-dystroglycan, no association between the gold label marking dystrophin and that marking spectrin was observed. Our freeze-fracture cytochemical results suggest that the two membrane skeletal networks formed by dystrophin and spectrin in cardiac muscle are independently organized, implying that whatever overlap of function (e.g., in structural support to the plasma membrane) may exist between them, the two systems may each have additional distinctive roles.     

”In vivo cryotechnique” in combination with replica immunoelectron microscopy for caveolin in smooth muscle cells

A novel ”in vivo cryotechnique” with replica immunoelectron microscopy was developed for detecting caveolin localization on replica membranes prepared directly from living smooth muscle cells. After quick-freezing mouse duodenal walls by our ”in vivo cryotechnique”, the specimens were prepared for freeze-fracture and deep-etch replica membranes. Then they were treated with 5% SDS and 0.5% collagenase to keep some antigens on the replica membranes. The immunogold method could be used to clarify the localization of the caveolin antigen in relation to three-dimensional ultrastructures of living smooth muscle cells. Our new cryotechnique can provide native organization of functional molecules in living cells. Accepted: 7 October 1999     

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.     

Characterization of the performance of a 200-kV field emission gun for cryo-electron microscopy of biological molecules

The value of an electron microscope equipped with a field emission gun (FEG) was first revealed in materials science applications. More recently, the FEG has played a crucial role in breaking the 10A barrier in single-particle reconstructions of frozen hydrated biological molecules. The standard high-resolution performance tests for electron microscopes are made close to focus, at several hundreds of A underfocus at a magnification of 500,000x or more. While this is appropriate for materials science specimens, it is not suitable for observing frozen hydrated biological specimens with which the optimum underfocus is of the order of 1 micron or so and the magnification is limited by radiation damage to roughly 30,000 to 60,000x. Thus, in order to access the performance of a cryo-electron microscope for high-resolution 3D electron microscopy of biological molecules, additional tests are necessary. We present here resolution tests of a 200-kV FEG using frozen hydrated virus suspensions. The extent and amplitude of the contrast transfer function are used as a test of the performance. We propose that small spherical viruses close to 300A in diameter, such as the picornaviruses or phages, make good specimens for testing the performance of an electron microscope in cryo-mode.     

X-ray scattering with momentum transfer in the plane of membrane. Application to gramicidin organization.

We demonstrate a technique for measuring x-ray (or neutron) scattering with the momentum transfer confined in the plane of membrane, for the purpose of studying lateral organization of proteins and peptides in membrane. Unlike freeze-fracture electron microscopy or atomic force microscopy which requires the membrane to be frozen or fixed, in-plane x-ray scattering can be performed with the membrane maintained in the liquid crystalline state. As an example, the controversial question of whether gramicidin forms aggregates in membrane was investigated. We used dilauroylphosphatidylcholine (DLPC) bilayers containing gramicidin in the molar ratio of 10:1. Very clear scattering curves reflecting gramicidin channel-channel correlation were obtained, even for the sample containing no heavy atoms. Thallium ions bound to gramicidin channels merely increase the magnitude of the scattering curve. Analysis of the data shows that the channels were randomly distributed in the membrane, similar to a computer simulation of freely moving disks in a plane. We suggest that oriented proteins may provide substantial x-ray contrast against the lipid background without requiring heavy-atom labeling. This should open up many possible new experiments.     

Detection of surface-bound ligands by freeze-fracture autoradiography

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

Label-fracture: a method for high resolution labeling of cell surfaces

We introduce here a technique, "label-fracture," that allows the observation of the distribution of a cytochemical label on a cell surface. Cell surfaces labeled with an electron-dense marker (colloidal gold) are freeze-fractured and the fracture faces are replicated by plantinum/carbon evaporation. The exoplasmic halves of the membrane, apparently stabilized by the deposition of the Pt/C replica, are washed in distilled water. The new method reveals the surface distribution of the label coincident with the Pt/C replica of the exoplasmic fracture face. Initial applications indicate high resolution (less than or equal to 15 nm) and exceedingly low background. "Label-fracture" provides extensive views of the distribution of the label on membrane surfaces while preserving cell shape and relating to the freeze-fracture morphology of exoplasmic fracture faces. The regionalization of wheat germ agglutinin receptors on the plasma membranes of boar sperm cells is illustrated. The method and the interpretation of its results are straightforward. Label-fracture is appropriate for routine use as a surface labeling technique.     

Characterization and crystal packing of three-dimensional bacteriorhodopsin crystals

The three-dimensional crystals of the integral membrane protein bacteriorhodopsin have been characterized by X-ray diffraction and freeze-fracture electron microscopy: the needle-like form A crystals belong to space group P 1 (pseudohexagonal) with seven molecules per crystallographic unit cell forming one turn of a non-crystallographic helix. The probable arrangement of the bacteriorhodopsin molecules is derived from freeze-fracture electron micrographs and chromophore orientation. Membrane-like structures are not present. The same helices of bacteriorhodopsin molecules found in crystal form A also make up the cube-like crystal form B. They are now arranged in all three mutually perpendicular directions. These cubes are always highly disordered, since the unit cell length corresponds to 6.7 molecules of the 7-fold helix. Very often, conversion of bacteriorhodopsin from the three-dimensional crystals into filamentous material occurs.