Freeze-fracture immunogold labeling

$: Freeze-fracture immunogold labeling$
Histochem Cell Biol (1996) 106:19-30 �9 Springer-Verlag 1996

M a r i a R o s a r i a Torr is i �9 P a t r i z i a M a n c i n i

Freeze-fracture immunogold labeling

Accepted: 2 May 1996

A b s t r a c t Several approaches have been developed to combine immunogold cytochemistry and freeze-fracture techniques. These methods are highly heterogeneous re- garding both the sequence of the procedural steps and the aspect of the resulting images. They imply immunolabeling either before or after freeze-fracture or even immunolabeling of platinum/carbon replicas of the freeze- fractured membranes, and have been used alternatively or in parallel to address different questions related to cell membrane structure, composition and dynamics or to intracellular membrane traffic. This review will briefly de- scribe these methods and report most of their immunogold cytochemical applications, with the aim of facilitat- ing selection of the most appropriate approach.

Introduction

Freeze-fracture technique was designed for the analysis of membrane structure. During freeze-fracture, cell membranes are split along their hydrophobic interior into two complementary halves, the outer or exoplasmic leaf-

M.R. Torrisi (~) �9 R Mancini Dipartimento di Medicina Sperimentale e Patologia Viale Regina Elena, 324 1-00161 Rome, Italy Tel. +39-6-4468450; Fax +39-6-4454820 e-mail trrsmr @flashnet.it

Fig. 1 A schematic view of the freeze-fracture process. During fracture, membranes (left) are split along their hydrophobic interior into two complementary leaflets: the exoplasmic outer leaflet (E/ace) and the protoplasmic inner leaflet (P face) (right). Transmembrane proteins may partition with the outer or the inner leaflet of the freeze-fractured membranes

let (E face) and the inner or protoplasmic leaflet (P face) (Fig. 1). Although lipids and peripheral proteins partition during fracture with the corresponding monolayer, integral and transmembrane proteins may partition with the outer or the inner membrane half. Platinum/carbon (Pt/C) replicas of freeze-fractured membranes provide views of the split membrane hydrophobic interior, where transmembrane proteins are believed to appear as intramembrane particles. Different methods have been therefore developed to analyze in a single image not only the ultrastructural features of the freeze-fractured membranes but also their composition and dynamics. The use of gold particles in lectin cytochemistry or in immunocytochemistry, because of their high electron density, en- abled easy visualization of the labeling on Pt/C replicas, and the efforts towards development of new methods in- creased considerably.

These methods can be separated in two groups: (a) techniques in which the immunolabeling is performed before freeze-fracture and replication, including the label-fracture and the fracture-flip techniques, and (b) different approaches in which the immunolabeling is performed on the replicas of the freeze-fractured membranes. Separate consideration is deserved by the fracture-label method, in which cells are labeled after freeze- fracture and samples may be observed in conventional thin sections. We will review here the methods in which freeze-fracture has been combined with immunogold la-

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beling and we will report studies where these methods have been applied to the analysis of membrane composition and dynamics.

Fracture-label

The fracture-label technique permits direct labeling of freeze-fractured plasma and intracellular membranes (Pinto da Silva 1984). Cells or tissues are fixed in glutaraldehyde, cryoprotected by 30% glycerol and frozen as in conventional freeze-fracture. The frozen samples are fractured in a glass or plastic container filled with liquid nitrogen by crushing with a glass or metal pestle to obtain small fragments, or using a scalpel to obtain larger fragments. The freeze-fractured samples are then thawed, gradually deglycerinated and cytochemically or immunocytochemically labeled. Two variants of the method have been developed: thin section fracture-label (Pinto da Silva et al. 1981b) and critical point drying fracture-label (Pinto da Silva et al. 1981a). In the first method, the fracture-labeled small samples are processed for conventional thin section electron microscopy, whereas in the second, larger samples are dried through the critical point and replicated by Pt/C evaporation. Al- though initially both variants were used in combination with lectin cytochemistry and frequently performed in parallel, critical point drying fracture-label has not been widely applied for immunolocalizations, probably because of the difficult interpretation of the fracture face replicas obtained, which appear altered and lacking typical ultrastructural details such as intramembrane particles. In contrast, thin section fracture-label provides full preservation of the cell ultrastructure, similar to that observed in conventional thin section images. Identification of the fracture plans and of the intracellular fractured membranes in thin sections is easy and the subsequent interpretation of the results is usually simple.

In fracture-label technique, either isolated cells or tissues can be used, in particular for labeling of intracellular components. When the purpose of the experiments requires labeling of the freeze-fractured plasma membranes, however, cells may be first embedded in a gel matrix of bovine serum albumin (BSA) cross-linked by glutaraldehyde, before freezing and fracturing (Fig. 2B-D). This procedure allows the gel to retain after fracture the outer exoplasmic leaflet of the plasma membrane and to prevent its loss during thawing (Fig. 2C). The gel matrix also occasionally permits one to maintain in close apposition and to simultaneously label the two complementary exoplasmic and protoplasmic fracture faces from the same plasma membrane (an event which has been called "crack") (Fig. 2B, D). On the other hand, fracture-label of isolated cells allows simultaneous comparison of the pattern and density of labeling on intracellular fractured membranes with those on the unfractured cell surfaces.

The ultrastructural observation of the freeze-fractured membrane leaflets shows interrupted bilayered segments

(Fig. 2B-D), instead of uninterrupted monolayers. This structural feature was interpreted as the result of post- fracture reorganization of the monolayers into bilayered structures occurring during thawing in an aqueous solu- tion. In fact, if the fractured samples are freeze-substitut- ed in osmium/acetone, a procedure which does not allow subsequent labeling, the fractured membrane halves appear as uninterrupted monolayers (Pinto da Silva et al. 1981b). On the other hand, these reorganization events are responsible for labeling on the exoplasmic faces of lectin binding sites or of epitopes originally exposed to the outer surface of the unfractured cells (Fig. 2A) and represent therefore a favorable process in fracture-label technique.

Immunogold thin section fracture-label has been applied mainly to address problems related to (a) the partition during fracture of transmembrane proteins and (b) the intracellular distribution of membrane components.

Partition of transmembrane proteins with the two fractured membrane leaflets may depend on the structural properties of the proteins themselves, as initially proposed from the fracture-label analysis of the partition on fracture of the two major glycoproteins, glycophorin and band 3, in human erythrocytes (Pinto da Silva and Torrisi 1982). The study showed that glycophorin, which is composed of a large extracellular portion, partitions preferentially with the outer exoplasmic face of the freeze- fractured membrane, whereas band 3, which is exposed mainly to the cytoplasmic side of the membrane and is associated with the cytoskeleton, partitions preferentially with the inner protoplasmic face. Subsequent studies on other cell systems, however, revealed that the depen- dence of behavior on fracture on protein structure cannot be considered a general rule and that stable or dynamic interactions of the transmembrane proteins with other membrane components or with membrane-associated elements may be determinant in this process. Most of the lymphocyte antigens we studied (CD8, HLA) partitioned during fracture with the exoplasmic face (Fig. 2B, D) (Mancini et al. 1994; Torrisi and Pavan 1993), but CD4, despite its structural similarity to glycophorin, was mostly associated with the inner protoplasmic membrane half (Pavan et al. 1989a). When CD8 antigens were cross-

Fig. 2A-D Surface immunolabeling (A) and fracture immunolabeling (B, C) of CD8 antigens on human lymphocytes. The immunogold labeling of CD8 molecules, performed with anti-CD8 antibodies and protein A-colloidal gold, is dense and uniform over the unfractured cell surface (A) of a CD8+ T lymphocyte, whereas platelets and CD8 lymphocytes appear unlabeled. In B, complementary protoplasmic (P) and exoplasmic (E) faces of a fracture- labeled plasma membrane of a CD8 + lymphocyte, where gold particles are confined to the E face and the P face appears unlabeled. E faces from freeze-fractured plasma membranes of CD8 lymphocytes are unlabeled (C). In D, fracture-immunolabeling of HLA antigens on human lymphocytes: the gold immunolabeling, performed with anti-HLA antibodies and protein A-colloidal gold, is distributed mainly on the E faces. Nu nucleus. A x16000, B x24000, C x33000, D x21000; bars" 1 ]am. B and C reproduced from Mancini et al. (1994); Verlag; D reproduced from Torrisi and Pavan (1993) by copyright permission of John Libbey Eurotext

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linked by incubation with specific antibodies at 37 ~ C to induce patching and capping, a process which is known to induce new interactions of the antigens with the underlying cytoskeleton, the partition of CD8 on fracture became protoplasmic (Mancini et al. 1994). In transfected cells, expressing only exogenous CD8 molecules at the cell surface, the capping process was not efficiently induced by cross-linking antibodies and the partition on fracture of CD8 remained exoplasmic, probably as a consequence of lack of interaction with the cytoskeleton (Mancini et al. 1994). All these results on fracture-labeled human lymphocyte plasma membranes seemed to confirm that interactions, more than structural properties, are responsible for partition on fracture with the protoplasmic faces, and extended the initial observation of heterogeneous expression of transmembrane sialoglyco- proteins on the inner membrane half of freeze-fractured lymphocytes (Torrisi and Pinto da Silva 1982). Consis- tent with the above observations, stable association of the protein dystrophin in skeletal muscle cells with the cytoskeleton appeared responsible for the association of the protein with the protoplasmic face of the fi'acture-labeled plasma membranes (Squarzoni et al. 1992).

Further support for the hypothesis that dynamic interactions can modify the behavior of transmembrane proteins on fracture came from a study on epidermal growth factor receptors (EGFRs) (Lotti et al. 1991), which were shown to modify their partition from exoplasmic to protoplasmic when activated by the EGF. Since this change in partition was more evident after receptor clustering at 37 ~ C, induced either by the ligand or by anti-receptor antibodies, it may reflect receptor cross-linking and interactions among the cytosolic tails.

In infected cells, interaction of the viral envelope proteins with the underlying nucleocapsids at the sites of budding was shown to be responsible for their behavior on fracture (Torrisi and Pavan 1995). In cells infected with Sindbis virus, an RNA enveloped virus used as a model system in membrane cell biology, the envelope viral glycoproteins were seen to partition with the exoplasmic faces of all intracellular membranes during their processing and transport to the cell surface; they acquired a protoplasmic partition only at the cell plasma membrane, because of interaction with the nucleocapsid during viral budding (Torrisi and Bonatti 1985). If the Sindbis virus proteins were expressed on the cell surface by transfection or following implantation by low-pH-mediated fu- sion, i.e. in the absence of nucleocapsid anchorage, their behavior on fracture was exoplasmic (Torrisi et al. 1990). The role played by nucleocapsid/envelope protein interaction in determining changes in partition on fracture of the viral transmembrane proteins seems to represent a general rule, followed also when budding occurs at intracellular membranes. In cells producing Epstein-Barr virus (EBV), where the envelope of the herpesvirus is acquired at the inner nuclear membrane, the major envelope glycoprotein was seen to cluster on budding figures and to partition with the protoplasmic face of the freeze- fractured inner nuclear membranes where nucleocapsids

were visible underneath the fractured leaflet (Torrisi et al. 1989b).

The second useful advantage of fracture-label technique is its possible application to the analysis of intracellular membrane components, approaching problems related to their distribution, compartmentalization and transport (Torrisi and Pinto da Silva 1984; Torrisi et al. 1989a). The method, in fact, exposes large areas of the intracellular membranes to the immunolabeling, providing full accessibility of the antigens; in the meantime, it preserves well the cellular ultrastructure and permits easy identification of the labeled organelles. In Sindbis virus-infected cells, immunogold fracture label was used to study the intracellular localization of the viral envelope glycoproteins, which are synthesized, processed and transported in large amounts to the cells surface, following the cellular biosynthetic and exocytic pathway. Im- munolabeling with specific antibodies and protein A- gold was found intracellularly on freeze-fractured endo- plasmic reticulum, outer nuclear membranes and Golgi cisternae (Torrisi and Bonatti 1985). The use of fracture- label technique in that study allowed us to observe an interesting finding: the presence of viral envelope glycoproteins on the inner nuclear membranes, a site not sup- posed to be involved in intracellular membrane traffic. With further fracture-label experiments, we were able to demonstrate that the viral glycoproteins may freely dif- fuse to and from the inner nuclear membrane, probably through the nuclear pore complexes (Torrisi et al. 1987).

The possibility, in fracture-label, to expose inner nuclear membranes to the immunolabeling was helpful in several studies on herpesvirus-infected cells, since these viruses acquire their envelope by budding at the inner nuclear membranes. Results obtained by parallel application of lectin cytochemistry and immunogold cytochemistry in fracture-labeled EBV-producing cells (Torrisi et al. 1989b) and herpes simplex type 1 (HSV-1)-infected cells (TolTisi et al. 1992) demonstrated that the two her- pesviruses acquire at the inner nuclear membranes an envelope similar in composition to that of extracellular virions, but containing immature type of proteins. In addi-

Fig. 3A-F Fracture-label of CD8-transfected epithelial cells (FRT-U10 cells, Mancini et al. 1994), showing examples of CD8 molecules on freeze-fractured intracellular membranes. Positive immunolabeling with anti-CD8 monoclonal antibody and protein A-colloidal gold is found on the exoplasmic faces of the freeze- fractured plasma membranes (A, B, D), and on Golgi cross-fractured (E) or freeze-fractured cisternae (F). Protoplasmic (A) and exoplasmic (B) faces of the freeze-fractured inner nuclear membranes are unlabeled. Freeze-fractured mitochondrium membranes appear also unlabeled (C). In E, the immunolabeling, penetrated inside the cross fractured cisternae, is dense on the inner surface of the dilatated rims (from E Mancini, M.C. Pascale, S. Bonatti, M.R. Torrisi, unpublished observations). P Protoplasmic face of freeze-fractured plasma membrane, E exoplasmic face of freeze- fractured plasma membrane, INp protoplasmic face of freeze-fractured inner nuclear membrane, 1Ne exoplasmic face of freeze-fractured inner nuclear membrane, Nu nucleus, M mitochondrium, G Golgi. A x14000, B x20000, C x31000, D x26000, E x37000, F x58000; bars A, B 1 ~tm, C-F 0.5 gm

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tion, in the herpesvirus-infected cell system, the pathway followed by the viral proteins and the virions for their intracellular transport and maturation is still debated. Frac- ture-label experiments demonstrated that, during intracellular transport of the viruses, the envelope glycoproteins become glycosylated to intermediate forms and finally to mature forms (Di Lazzaro et al. 1995), implying transport of the virions through the Golgi complex. In Vero cells infected with a mutant HSV-1, deleted in the UL20 protein, the immunogold fracture-label method revealed that the viral glycoproteins are present on the nuclear membranes, but are not transported to the cell surface, sug- gesting a role for the UL20 protein in the transport of viral glycoproteins to the cell plasma membranes and in the exit of the virions from the cell (Avitabile et al. 1994).

Fracture-label was also used by another group to study the intracellular distribution of opsin in rod photoreceptor membranes of Xenopus laevis and mouse reti- nas: immunolabeling revealed localization of opsin on rod outer segments, and its transport from rod inner to outer segments, as a mechanism occurring during disc assembly (Defoe and Besharse 1985; Besharse and Wet- zel 1995). An interesting application of fracture-label, although not involving the immunolabeling approach, was reported for the localization and quantitation of phospholipids in fractured tissues using enzyme-gold cytochemistry (Coulombe etal. 1988): phospholipase A2, ad- sorbed to colloidal gold, specifically labeled the glyc- erophospholipids on intracellular membranes and permitted comparison of the labeling density over different membrane compartments.

In Fig. 3, examples of immunolabeling of CD8 molecules on freeze-fractured Fisher rat thyroid epithelial cells (FRT-UI0 cells) expressing by transfection CD8 protein are shown. As reported above (Mancini et al. 1994), the labeling on the freeze-fractured plasma membranes was confined to the exoplasmic face. The intracellular freeze-fractured membranes were differentially immunolabeled by the anti-CD8 monoclonal antibody used, which recognizes mainly the mature forms of the protein after processing through the Golgi apparatus (R Mancini, M.C. Pascale, S. Bonatti, M.R. Torrisi, unpublished results). The gallery of images shown in Fig. 3 points out how, in fracture-label samples, the intracellular ultrastructure is well preserved and provides examples of different intracellular membrane fractured faces and fractured organelles.

Label-fracture

The label-fracture technique represents an ideal method to study at high resolution the distribution of surface molecules, such as antigens or receptors, on the cell plasma membranes (Pinto da Silva and Kan 1984). The technique, although limited to the exoplasmic outer leaflet of the freeze-fractured plasma membrane, permits correla- tion, in a single view, of the distribution of the surface labeled membrane components and that of typical freeze- fracture morphological features such as the intramem-

Fig. 4A-D Label-fracture. A Isolated cells are gold immunolabeled and frozen. B Freeze-fracture splits the cell plasma membranes into exoplasmic outer and protoplasmic inner halves. C Replicas of the freeze-fractured membranes are obtained by platinum (Pt) and carbon (C) evaporation. D Samples are thawed and washed in distilled water. Exoplasmic membrane halves and entire cells with protoplasmic membrane halves remain attached to the Pt/C replicas. Replicas are then picked up on grids, dried and observed at TEM. (Modified from Pinto da Silva and Kan 1984)

brane particles (IMPs). The comparison of the distribution of gold immunolabeling and of IMPs and their possible coincidence can be better visualized by experimen- tally inducing redistribution and clustering of either the surface labeled components or of the IMPs. In this technique (Fig. 4), the cells are first immunolabeled with specific antibodies directed against surface exposed antigens, followed by protein A-gold or secondary antibody-

gold conjugated. The size of gold particles should be suf- ficiently large to appear clearly visible behind a Pt/C cast (usually particles of at least 10 nm in diameter are used). A good advantage of this method is that the immunolabeling can be performed also on unfixed cells, preserving the best conditions for optimal antigenicity and labeling efficiency; in addition, double or triple labeling proce- dures can also be easily applied. After immunolabeling, cells are fixed in glutaraldehyde, impregnated in glycerol as a cryoprotectant and frozen. Cells are freeze-fractured in a freeze-fracture device and the fracture faces replicated by Pt/C evaporation as in conventional freeze-fracture technique. Instead of being digested with acids or deter- gents, replicas are floated in distilled water, picked up with Formvar-coated grids and observed in a transmis- sion electron microscope. This procedure allows the labeled outer exoplasmic leaflet of the plasma membrane to remain attached to the replica. Immunolabeling in the resulting image is underneath the fracture face and, therefore, gold particles are not shadowed.

The label-fracture method was initially applied to map lectin binding sites on human erythrocyte and leu- kocyte membranes (Pinto da Silva and Kan 1984) and on boar sperm cell surfaces (Aguas and Pinto da Silva 1984; Pinto da Silva 1987). An interesting approach, combin- ing autoradiography and label-fracture method, was used to localize radioactive-ligand binding to surface receptors (Carpentier et al. 1985). Our group has mostly applied the label-fracture technique to analyze the dynamics during patching and capping of lymphocyte transmembrane proteins. We first analyzed the clustering of CD3 and CD4 human lymphocyte antigens (Pavan et al. 1989a) and the possible aggregation of IMPs. Surface immunolabeling was performed using specific monoclo- hal antibodies directed against the lymphocyte antigens, followed by protein A-colloidal gold conjugates. To demonstrate that the transmembrane proteins bearing the antigens correspond to IMPs, we induced a generalized aggregation of IMPs by treatment of unfixed lymphocytes with 30% glycerol. In these samples, the immunolabeling was confined to the particle aggregates, showing that the CD4 and CD3 transmembrane proteins correspond to IMPs. Patching and capping of the antigens was then induced by treatment with the corresponding antibodies at 37 ~ C, but it was not accompanied by a parallel redistribution of IMPs. These observations have been interpreted as the consequence of a highly dynamic process in which migration to the capping area of IMPs corresponding to the antigens implies exclusion from that area of IMPs corresponding to other membrane proteins not involved in the capping phenomenon. These dynamic events would be responsible for maintaining a structural equilibrium in the plane of the plasma membrane. These results have been confirmed by further applications of the same method to follow the clustering of other lymphocyte antigens, such as HLA and LFA-1 molecules (Pavan et al. 1989b, 1992a) (Figs. 5, 6). Both the timing and sequence of antigen migration, first to small patches and then to larger patches and to a final single cap, was comparable for all antigens analyzed. The capping pro-

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Fig. 5A, B Immunogold label-fracture of human lymphocytes. A Distribution of immunolabeled HLA antigens on the cell surface is uniform in pre-fixed cells. Intramembrane particles (IMPs) on the exoplasmic fracture face appear also uniformly distributed. B Patching of HLA antigens, induced by incubation with specific antibodies at 37 ~ C for 10 min, shows large clusters of gold particles on the cell surface. IMPs on the exoplasmic face appear uniformly distributed. A x21000, B xl8000; bars' 1 gm. Reproduced from Torrisi and Pavan (1993) by copyright permission of John Libbey Eurotext

cess was more efficient when LFA-1 molecules were linked to the cytoskeleton upon phorbol ester treatment (Pavan et al. 1992a), a finding in favor of the participa- tion of cytoskeletal elements in the regulation of this process. The label-fracture images in all these conditions

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Fig. 6 Stereo pair views of an immunogold label-fractured human lymphocyte. Capping of LFA-1 molecules is induced by treatment with anti-CD18 monoclonal antibody at 37~ for 20 rain. The gold particles are concentrated into a single cap. IMPs are not concentrated in the capping area, but appear uniformly distributed on the exoplasmic fracture face. x30000; bar 0.5 gm. Reproduced from Pavan et al. (1992)

on human erythrocytes revealed a clustered distribution of the receptors and permitted quantitation of the number of clusters per cell and the size of the single cluster (Chevalier and Kazatchkine 1989). Successively, the method allowed comparison of the CR1 clusters observed on erythrocytes with those present on the surface of resting or stimulated polymorphonuclear leukocytes, and the results were discussed in light of the role played by CR1 in the immune response (Paccaud et al. 1990). Finally, a more recent application of immunogold label- fracture to locate surface receptors is described in two studies on GABAA receptors. In the first, the distribution of the receptors in various neuronal cell types and in cells transfected with different receptor subunits was compared (Caruncho et al. 1993); in the second, double immunolabeling using gold particles of different sizes was used to analyze the subunit composition of the receptor (Caruncho and Costa 1994).

A variation of the label-fracture method has been in- troduced for the analysis of the ultrastructure and composition of isolated lens fiber junctions (Zampighi et al. 1989). Junctions were immunolabeled, adhered on a glass surface and covered in sandwich with a copper hat before freeze-fracture and replication.

confirmed that uniform distribution of IMPs obtained was a result of re-equilibrium among structural components of the plasma membranes.

A different situation was found when the label-fracture technique was used in Sindbis virus-infected cells to study the dynamics of viral envelope glycoproteins during the budding of the virus at the cell surface (Pavan et al. 1992b). In fact, IMPs appeared concentrated at the budding sites, and the particle aggregates corresponded to the clustering of the viral glycoproteins, as shown by specific gold immunolabel with antibodies to the envelope proteins. This study provided an additional example of the potentiality offered by label-fracture to relate the distribution of membrane components with ultrastructural features of the plasma membranes. This approach was also used for another viral system, in a study on measles virus-infected cells (Rutter and Hohenberg 1993), where label fracture was applied to isolated plasma membranes, instead of intact cells, to extend the observation on the protoplasmic fracture faces and to compare their ultrastructure with immunolabeling of viral proteins at the inner cytoplasmic surface.

Several cell surface receptors have also been studied by the label fracture technique. The method was used on cell monolayers (Boonstra et al. 1985) to detect gold-immunolabeled EGFRs on the cell surface of a human epithelial cell line: comparison of the results with those obtained in parallel with the surface-replication method in- dicates that label-fracture is the ideal technique for quan- titative analysis of the planar distribution of the receptors on the cell surfaces. Later, the same approach was used to follow the redistribution of EGFRs induced by the ligand binding (Rijken et al. 1993). Analysis by label-fracture of the localization of complement receptors (CR1)

Fracture-flip

The fracture-flip method, although requiring a freeze- fracture procedure, permits observation of the real surface of the cell, which becomes visible by inversion (flipping) of the exoplasmic fracture face (Fig. 7). In fact, fracture-flip can be considered a variant of the label-fracture approach, since the initial steps are identical to those of label-fracture preparations. At variance with label-fracture, it requires after freeze-fracture coating of the exoplasmic face only with carbon. The replicas are then floated in distilled water as for label-fracture, picked up from the top with a formvar-coated grid, in- verted (flipped) and shadowed at room temperature by platinum evaporation. The resulting replica provides high-resolution views of the cell surface ultrastructure, revealing details such as surface particles, subparticles and rugosities (Andersson-Forsman and Pinto da Silva 1988a). If cells are immunolabeled, the gold particles are on the replicated cell surface and appear therefore shadowed.

We first applied immunogold fracture-flip to analyze the dynamics of surface particles during patching/capping of lymphocyte antigens (CD4 and HLA molecules) (Pavan et al. 1990), as an extension of our previous studies on capping (Fig. 8). Sequential clustering of the antigens obtained by incubation with specific antibodies at 37 ~ C was performed similarity.to the experiments for label-fracture described above. Since gold immunolabeling obscures the ultrastructural details of the bound antigens, we compared cells treated with the antibodies followed, or not, by protein A-gold labeling. In the resulting images, either from gold-labeled or unlabeled cells, the patching and capping areas appeared as platforms different in

Fig. 7A-D Immunogold fracture-flip. The initial steps of the method are identical to those drawn schematically in Fig. 4A, B. A After immunolabeling and freeze-fracture, the exoplasmic and protoplasmic fracture faces are stabilized by carbon (C) evaporation. B Samples are thawed and washed in distilled water as in label-fracture. The immunolabeled exoplasmic membrane halves remain attached to the C replicas. Replicas are picked on a grid by the top and dried. C Flipping or inversion of the grid exposes the immunolabeled outer cell surface. D Platinum (Pt) deposition pro- duces high-resolution casts, which are viewed at TEM. (Modified from Andersson-Forsman and Pinto da Silva 1988a)

size, depending on the extent of the clustering and on the 37~ incubation time. In these experiments we were able to quantitate the antigen concentration in the patches/caps and to confirm that the capping process is reached by progressive merging of small patches into

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larger patches and finally into a single final cap. More- over, incubation with glycerol induced not only aggregation of IMPs on the exoplasmic fracture plan, as reported above, but also aggregation of surface particles (SPs) on the flipped outer surface. Since both particle aggregates could be labeled by antibodies directed against the transmembrane lymphocyte antigens, we concluded that the SPs visible in fracture-flip images represent the surface protrusions of transmembrane proteins which appeared as IMPs on the exoplasmic fractured faces. The capping process was also followed for LFA-1 antigens on fracture-flipped cells (Pavan et al. 1992a), showing that SPs were uniformly distributed over non-capped areas at all times of warming to 37 ~ C, as observed for the other antigens (Fig. 8). Here, however, our purpose was to ascer- tain whether a linkage of the LFA-1 antigens to the cytoskeletal talin, which resulted in a more efficient capping process, could modify the distribution of surface particles over the non-capped areas: our observations indicat- ed that the interaction of the antigens with the cytoskeleton, although affecting the antigen dynamics, does not alter the membrane ultrastructure, providing further evi- dence for a structural re-equilibrium of membrane components on the plane of the cell plasma membranes.

The fracture-flip technique also appears ideal for studies directed to other dynamic events occurring at the cell surface, such as the budding of viruses on the plasma membranes of the infected cells (Pavan et al. 1992b). We have applied this method in combination with immunogold labeling to analyze, in cells infected with Sindbis virus, the concentration of viral glycoproteins at the budding sites and to prove the exclusion of cellular proteins from these sites. As observed in parallel label-fractured preparations, the viral glycoproteins were localized at the budding sites, where they appeared highly concentrated. This protein concentration corresponded in unlabeled samples to a clear aggregation of SPs on the budding figures. Therefore, at variance with the patching/capping phenomenon, where concentration of the antigens is not accompanied by a coincident aggregation of membrane particles, during viral budding the concentration of viral glycoproteins involves aggregation of membrane particles, probably reflecting immobilization of the viral envelope proteins by interaction of their cytosolic tails with the underlying nucleocapsids. In addition, parallel immunolabeling of CD8 proteins, expressed on the plasma membrane of the infected cells, revealed exclusion of cellular proteins from the viral budding sites.

Fracture-flip and immunolabeling have been also combined to visualize the binding of bacterial endotoxins to the surface of macrophages, a process which leads to macrophage activation (Risco and Pinto da Silva 1993).

If the fracture-flip method is applied to cells growing in monolayer on glass coverslips or cells capable of ad- hering or spreading on a substratum, the flipped outer surfaces may correspond either to the dorsal apical surface or to the ventral basal surface of the fractured cells (Fujimoto and Pinto da Silva 1988). The cells are covered with a gold specimen carrier before freezing and

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Fig. 8A-C Stereo views ofim- munogold fracture-flipped human lymphocytes. A Distribu- tion of LFA-I molecules on pre-fixed cells, immunolabeled with anti-CD 18 monoclonal antibody and protein A-colloidal gold. The gold particles appear uniformly distributed on the cell surface. Ultrastructural details such as surface particles of the plasma membranes are clearly visible. B, C Capping of HLA antigens, induced by incubation with anti-HLA I class antibodies at 37 ~ C for 20 rain, reveals high concentration of the gold particles in a single cap. The distribution of surface particles appears unaltered. Note that in fracture-flip images the immunogold particles are shadowed, unlike those observed in label-fracture preparations. A x13000; B x17000; C x22000; bars 1 gin. A Re- produced from Pavan et al. (1992); B reproduced from Pa- van et al. (1990) by copyright permission of The Company of Biologists Limited; C reproduced from Torrisi and Pavan (1993) by copyright permission of John Libbey Eurotext

fracturing. This method has been used to observe the formation of coated pits on the adherent surface of spreading macrophages (Fujimoto and Pinto da Silva 1988), but it can be generally applied to compare surface domains of polarized cells ultrastructurally and immunocytochemically at high resolution.

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Acknowledgements We thank Dr Angelo Del Nero and Giuseppe Lucania for the excellent graphic and photographic work and edi- torial assistance. The funding support of grants from AIRC and CNR (PF ACRO), Italy, is acknowledged.

References

Freeze-fracture replica immunolabeling

This heterogeneous group of methods has in common the ability 1o label, after freeze-fracture, the fracture face replicas. However, it is important to point out that, during fracture, t ransmembrane proteins partition with the two leaflets and, therefore, the resulting fracture faces do not contain the entire set of proteins present on the unfractured inner or outer surfaces. Therefore, labeling after fracture does not warrant, as a general rule, staining of the designated membrane component.

The first technique of this group was proposed as a variant of label-fracture, based on the assumption that in label-fracture preparations the exoplasmic faces remain undigested and attached to the replica and that therefore the outer surface of the plasma membrane is exposed for possible immunolabeling (Andersson-Forsman and Pinto da Silva 1988b). Briefly, the replicas of the exoplasmic faces, after washing in distilled water, are picked up with a grid and labeled onto drops of the immunocytochemical reagents, like for staining of thin sections. Due to non-specific adsorption of colloidal gold to the Pt/C cast, the resulting images show a very high density of gold particles between the exoplasinic faces. Specificity of the immunolabeling must therefore be judged carefully, comparing the label on the cell surfaces in experiment and control samples.

The method of immunolabeling freeze-fracture replicas has been used to localize MP70 lens membrane protein on the junctional plaques of lens fibers (Gruijters et al. 1987), where the method could be easily applied because of the thinness of this type of samples. Recently a new approach has been proposed for the simultaneous analysis of structural organization and composition of in- tercellular junctions (Fujimoto 1995): tissue slices are freeze-fractured, replicated and digested in sodium dode- cylsulfate (SDS) before immunolabeling. Interestingly, SDS treatment dissolves all unfractured cell components but it cannot digest the fractured faces stabilized by the Pt/C cast; this makes it possible to label not only outer cell surfaces behind the exoplasmic faces but also cytoplasmic surfaces behind the protoplasmic faces. The method can also be applied to isolated cells, but it may be limited by the partition during fracture of integral membrane proteins, as discussed above.

In conclusion, the capacity to immunolabel intracellular and/or cell surface components specifically and efficiently renders freeze-fracture a powerful approach with general application in electron microscopy.

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Freeze-fracture immunogold labeling

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