Recent advances in freeze-fracture electron microscop: the replica immunolabeling technique

Freeze-fracture electron microscopy is a technique for examining the ultrastructure of rapidly frozen biological samples by transmission electron microscopy. Of a range of approaches to freeze-fracture cytochemistry that have been developed and tried the most successful is the technique termed freeze-fracture replica immunogold labeling (FRIL). In this technique samples are frozen fractured and replicated with platinum-carbon as in standard freeze fracture and then carefully treated with sodium dodecylsulphate to remove all the biological material except a fine layer of molecules attached to the replica itself. Immunogold labeling of these molecules permits their distribution to be seen superimposed upon high resolution planar views of membrane structure. Examples of how this technique has contributed to our understanding of lipid droplet biogenesis and function are discussed.     

Immunogold localization of basement membrane molecules in rat retinal capillaries

Vascular basement membrane contains laminin, fibronectin, proteoglycan and collagens. These molecules have been identified in various tissues by immunolabeling methods and biochemical analyses. We have previously localized laminin, fibronectin and type IV collagen to the basement membrane of rat retinal vessels at the ultrastructural level using an immunoperoxidase method. In this study, we use an immunogold method to re-examine the distribution of these molecules and also to study the localization of heparan sulfate proteoglycan and types I, III and V collagen in the retinal capillary basement membrane. Gold labeling for laminin, type IV collagen and proteoglycan were found diffusely on the basement membrane of the endothelium and pericyte, while that for fibronectin and type V collagen was spotty and variable and that for types I and III collagen was negligible. The segment of basement membrane between the endothelial cell and pericyte appeared less reactive to anti-laminin and anti-type IV collagen than the membrane between the pericyte and perivascular neuroretina. The immunogold method may be useful in quantitative studies of thickened basement membranes under abnormal conditions.     

Effects of bacteriophage fd infection on Escherichia coli HB11 envelope: a morphological and biochemical study.

Phage fd-infected host bacteria revealed three characteristic changes in their envelope. (i) The preferred cleavage plane during freeze-fracturing shifted from the inner to the outer membrane (OM). (ii) The total lipids of the OM of the infected cells increased by 25% without major alterations in the relative concentration of phospholipids. We propose that such an increase would to some extent contribute to the change in the freeze-fracture behavior of the OM; however, additional factors will have to play a role in the apparent fracture resistance of the inner membrane. (iii) Ultrathin sectioning and immunolabeling methods revealed that extrusion of fd phages takes place at membrane adhesion sites of the infected cells.     

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.     

The shape of caveolae is omega-like after glutaraldehyde fixation and cup-like after cryofixation

Caveolae were defined as flask- or omega-shaped plasma membrane invaginations, abundant in adipocytes, fibroblasts, endothelial and smooth muscle cells. The major protein component of caveolar membranes is an integral membrane protein named caveolin. We compared the freeze-fracture behavior of caveolae in glutaraldehyde-fixed and cryofixed mouse fibroblast cells and found distinct differences. In glutaraldehyde-fixed cells almost all caveolae were cross-fractured through their pore and only very few caveolar membranes were membrane-fractured. We found the reverse situation in rapid frozen cells without any chemical fixation where most of the caveolae were membrane-fractured, showing different degrees of invagination from nearly flat to deeply invaginated. In ultrathin sections of glutaraldehyde-fixed heart endothelial cells, caveolae exhibit the well known omega-like shape. In high-pressure frozen, freeze-substituted and low temperature embedded heart endothelial cells, the caveolae frequently exhibit a cup-like shape without any constriction or pore. The cup-like caveolar shape could also be shown by tilt series analysis of freeze-fracture replicas obtained from cryofixed cells. Freeze-fracture immunolabeling of caveolin-1 revealed a lateral belt-like caveolin alignment. These findings point out that the constricted “neck” region of caveolae in most cases is an effect that is caused and intensified by the glutaraldehyde fixation. Our data indicate that caveolae in vivo show all degrees of invagination from nearly flat via cup-like depressed to in a few cases omega-like.     

Uptake and subcellular distribution of Escherichia coli lipopolysaccharide by isolated rat type II pneumocytes

Treatment of isolated rat Type II pneumocytes with Escherichia coli lipopolysaccharide (LPS) induces a number of ultra-structural changes which become evident after 60 min of incubation. By using post-embedding immunolabeling methods and electron microscopy, we have followed the fate of LPS after different times of incubation. After an initial period of accumulation in the pneumocyte microvilli, the LPS molecules enter the cytoplasm, forming discrete patches which are dispersed in some areas. After longer incubation times, LPS localize in condensed chromatin-free areas inside the nuclei. LPS micelles were visualized after freeze-fracture and compared with the LPS-labeled membrane areas, showing that LPS micelles aggregate in particular membrane zones. The sugar-specific staining in microvilli areas, where Maclura pomifera agglutinin (MPA)-gold particles bind, indicates the presence of galactose derivatives in these membrane structures. Pre-treatment of pneumocytes with LPS inhibited the MPA-gold labeling, suggesting a relation between the MPA receptor and a possible LPS receptor. Finally, double immunolabeling experiments indicated an apparent LPS-tubulin association in some particular membrane regions, which could not be observed when LPS and actin were co-localized.     

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.     

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.     

Subcellular localization of secretogranin II and synaptophysin by immunoelectron microscopy in differentiated hypothalamic neurons in culture

Secretogranin II (SgII), a tyrosine-sulfated secretory protein, is a widespread component of endocrine and neuronal cells. In the present study we used mouse hypothalamic neurons differentiated in culture and studied the subcellular localization of SgII by two methods, i.e., by the use of immunoperoxidase or immunogold electron microscopy. By immunoperoxidase labeling, SgII was mainly detected in the matrix of large dense-core vesicles (LDCVs). In addition, usually in nerve terminals containing LDCVs, peroxidase reaction product was also found in association with the membrane of small synaptic vesicles (SSVs). By immunogold labeling, SgII was detected only in the matrix of LDCVs. We also compared the localization of SgII and synaptophysin (SY), an integral membrane protein of SSVs, by double labeling, using a combination of pre-embedding immunogold and -peroxidase techniques for SgII and SY, respectively. In perikarya, SgII-positive LDCVs were observed in the vicinity of the Golgi complex and scattered in the cytoplasm. In contrast, SY labeling was restricted to electron-translucent vesicles and tubular membranes in the Golgi area. Moreover, membrane structures positive for both SgII and SY were not found either in the Golgi zone or in other regions of the cytoplasm. In synaptic boutons, immunolabeling of LDCVs and SSVs with anti-SgII and anti-SY, respectively, was mutually exclusive. In summary, within the limitation of the methods used, our data are consistent with the notion that SgII and SY are segregated from each other on exit from the trans-Golgi network, than follow two distinct membrane traffic pathways, and that the presence of SgII on the membrane of some SSVs is due to endocytosis.     

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.     

Location of Ryanodine and Dihydropyridine Receptors in Frog Myocardium

Frog myocardium depends almost entirely on calcium entry from extracellular spaces for its beat-to-beat activation. Atrial myocardium additionally shows internal calcium release under certain conditions, but internal release in the ventricle is absent or very low. We have examined the content and distribution of the sarcoplasmic reticulum (SR) calcium release channels (ryanodine receptors, RyRs) and the surface membrane calcium channels (dihydropyridine receptors, DHPRs) in myocardium from the two atria and the ventricle of the frog heart using binding of radioactive ryanodine, immunolabeling of RyR and DHPR, and thin section and freeze-fracture electron microscopy. In cells from both types of chambers, the SR forms peripheral couplings and in both chambers peripheral couplings colocalize with clusters of DHPRs. However, although a low level of high affinity binding of ryanodine is detectable and RyRs are present in peripheral couplings of the atrium, the ventricle shows essentially no ryanodine binding and RyRs are not detectable either by electron microscopy or immunolabeling. The results are consistent with the lack of internal calcium release in the ventricle, and raise questions regarding the significance of DHPR at peripheral couplings in the absence of RyR. Interestingly, the free SR membrane in both heart chambers shows a low but equal density of intramembrane particles representing the Ca²⁺ ATPase.     

Immunogold localization of the NodC and NodA proteins of Rhizobium meliloti.

Monospecific, polyclonal antibodies to the nodC and nodA gene products of Rhizobium meliloti were used in combination with immunogold labeling and transmission electron microscopy to localize the NodC and NodA proteins in cultures of R. meliloti. Both NodC and NodA were detected in the cytoplasm and cell envelope in thin sections of free-living rhizobia treated with luteolin, a known inducer of nod gene expression; however, only NodC was detected on cell surfaces when immunolabeling was performed with intact induced cells. In view of biochemical data characterizing NodC as an outer membrane protein with a large extracellular domain, the pattern of immunolabeling on thin sections suggests that NodC is produced on free cytoplasmic ribosomes prior to assembly in the membrane. The pattern of NodA labeling on thin sections is consistent with biochemical data detecting NodA in both soluble and membrane fractions of NodA-overexpressing strains of R. meliloti.     

Membrane recycling after exocytosis: An ultrastructural study of cultured chromaffin cells

When exocytosis of granule contents is induced by nicotine stimulation, glycoprotein III (a chromaffin granule membrane constituent) is exposed on the surface of cultured chromaffin cells, where it may be labeled with an immunocytochemical tracer. The subsequent fate of this glycoprotein after endocytosis was followed at the ultrastructural level using immunogold methods and was analyzed by morphometry. After stimulation exocytosis membranes newly inserted into the plasma membrane labeled with gold particles for glycoprotein III were found to be endocytosed via coated vesicles and finally found in organelles devoid of chromogranin A, the major secretory granule protein. At intervals between 30 min and 24 h after cell stimulation and immunolabeling, most labeled structures were identified by two different morphological approaches as prelysosomes and lysosomes. In contrast with results obtained on freshly isolated chromaffin cells, it is thus concluded that in cultured cells granule membrane recycling into new granules does not occur. It is suggested that the fate of granule membrane endocytosed after cell stimulation may be influenced by the external conditions to which cells are previously exposed.     

Studies on the orientation of brush-border membrane vesicles.

Orientation of rat renal and intestinal brush-border membrane vesicles was studied with two independent methods: electron-microscopic freeze-fracture technique and immunological methods. With the freeze-fracture technique a distinct asymmetric distribution of particles on the two membrane fracture faces was demonstrated; this was used as a criterion for orientation of the isolated membrane vesicles. For the immunological approach the accessibility or inaccessibility of aminopeptidase M localized on the outer surface of the cell membrane to antibodies was used. With both methods we showed that the brush-border membrane vesicles isolated from rat kidney cortex and from rat small intestine for transport studies are predominantly orientated right-side out.     

Ultrastructure, histological distribution, and freeze-fracture immunocytochemistry of gap junctions in rat brain and spinal cord

The historical development of concepts of gap junctions as sites for electrical, ionic, and metabolic coupling is reviewed, from the initial discovery of gap junctions linking heart cells, to the current concepts that gap junctions represent 'electrotonic synapses' between neurons. The ultrastructure and immunocytochemistry of gap junctions in heart, brain, and spinal cord of adult rats is examined using conventional thin sections, negative staining, grid-mapped freeze-fracture replicas, and immunogold-labeled freeze-fracture replicas. We review evidence for neuronal gap junctions at 'mixed' (combined electrical and chemical) synapses throughout adult rat spinal cord. We also show immunogold labeling of connexin43 in astrocyte and ependymocyte gap junctions and of connexin32 in oligodendrocyte gap junctions. Ultrastructural and freeze-fracture immunocytochemical methods have provided for definitive determination of the number, size, histological distribution, and connexin composition of gap junctions between neurons in all regions of the central nervous systems of vertebrate species.     

Quantitative immunocytochemical study of blood-brain barrier glucose transporter (GLUT-1) in four regions of mouse brain.

Application of immunogold cytochemistry revealed polar (asymmetric) distribution of GLUT-1 in mouse brain microvascular endothelia, representing the anatomic site of the blood-brain barrier (BBB). This polarity was manifested by an approximately threefold higher immunolabeling density of the abluminal than the luminal plasma membrane of the endothelial cells. The immunoreaction for GLUT-1 in nonbarrier continuous (skeletal muscle) or fenestrated (brain circumventricular organs) microvascular endothelial cells was absent. In the choroid plexus, the basolateral plasmalemma of the epithelial cells was labeled more intensely than the vascular fenestrated endothelium. Addition of morphometry to the applied immunogold technique makes it possible for even subtle differences to be revealed in the density of immunolabeling for GLUT-1 in blood microvessels located in four brain regions. We found that the density of immunosignals in the microvessels supplying the cerebral cortex, hippocampus, and cerebellum was essentially similar, whereas in the olfactory bulb it was significantly lower. Asymmetric distribution of GLUT-1 in the endothelial plasma membranes presumably leads to a reduced concentration of glucose molecules in the endothelial cells compared to blood plasma and also secures their more rapid transport across the abluminal plasmalemma to the brain parenchyma.     

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.     

Rat intestinal galactoside-binding lectin L-36 functions as a structural protein in the superficial squamous cells of the esophageal epithelium

Using an affinity purified antibody raised against the RI-H fragment of rat intestinal lectin L-36, the latter protein has been identified within the esophageal epithelium by means of ultracryotomy followed by immunogold labeling. The epithelium consists of 4 morphologically distinct cell-types, namely, the basal, spiny, granular and squamous cells, and each of these exhibits a different immunolabeling pattern. The basal cells form a layer on the basal lamina, and in these a diffuse cytoplasmic staining is observed. This basal cell layer is overlaid by spiny cells that extend many cell processes into wide intercellular spaces. In these cells, immunogold particles are found only on small granular inclusions consisting of an electron-lucent homogeneous substance. The granular cells from a third layer over the spiny cells, and are characterized by a number of large granular inclusions with an electron-dense core rimmed by a less electron-dense substance. Immunogold labeling is found on these granules, both on the core and peripheral region. Squamous cell-types constitute the most superficial layer of the epithelium. They are without granular inclusions, and immunogold labeling is confined to the cytoplasmic surface of the thickened plasma membrane. These findings suggest that L-36 is produced in the basal cells as free cytosolic protein, then becomes progressively aggregated into the granular inclusions of the spiny and granular cells, and is eventually transferred onto the cytoplasmic surface of the squamous cell plasma membrane where it may interact with complementary glycoconjugate(s) located at this site. The membrane lining substance thus formed may play a role in stabilizing the squamous cell membranes, thereby maintaining the structural integrity of the epithelium against mechanical stress coming from the esophageal lumen.     

Direct immunogold labeling of connexins and aquaporin-4 in freeze-fracture replicas of liver, brain, and spinal cord: factors limiting quantitative analysis

Direct immunogold labeling and histological mapping of membrane proteins is demonstrated in Lexan-stabilized SDS-washed freeze-fracture replicas of complex tissues. Using rat brain and spinal cord as primary model systems and liver as a "control" tissue to identify preparation and labeling artifacts, we demonstrate the presence of connexin43 in freeze-fractured gap junctions of identified and mapped astrocytes and ependymocytes, and confirm the presence of connexin32 in freeze-fractured gap junctions in liver. In addition, the simultaneous double-labeling of dissimilar proteins (connexin43 and aquaporin-4) is demonstrated in gap junctions and square arrays, respectively, in the plasma membranes of astrocytes and ependymocytes. Finally, double-side shadowing and conventional staining methods are used to reveal the extent of biological material present at the time of labeling and to investigate the dynamics of membrane solubilization, the primary artifacts that occur during labeling, and several factors limiting quantitative analysis.     

Studies of human skin by cryofractographic and electron-microscopic methods

Ultrathin slices from the human abdominal skin and platinum-carbonic replicas, obtained by means of the freeze-fracture method have been studied electron microscopically. Transmembranous proteins are revealed on the membrane surface as membrane-bound particles with the diameter 5-7 nm. They mainly concentrate on the membrane protoplasmic surface. Certain difference is noted in distribution of tonofibrils in cytoplasm of the epidermal cells, when various methods for preparation of specimens to be investigated are used. Predominance of the freeze-fracture method comparing to the routine method of electron microscopy is discussed, since the specimens do not subjected to fixation, dehydration and resin saturation.