Localization of the ASV src gene product to the plasma membrane of transformed cells by electron microscopic immunocytochemistry

Cell, Vol. 18, 125-l

34, September

1979,

Copyright

0 1979

by MIT

Localization of the ASV WC Gene Product to the Plasma Membrane of Transformed Cells by Electron Microscopic lmmunocytochemistry Mark C. Willingham, Gilbert and Ira Pastan National Cancer Institute Bethesda, Maryland 20205

Jay

Summary The cellular location of the src gene product (p60”‘“) of the Schmidt-Ruppin strain of avian sarcoma virus has been determined by electron microscopic immunocytochemistry in Schmidt-Ruppin ASV-transformed NRK cells, and the amount of the protein in different regions of the cell has been quantified. The protein is concentrated on the inner surface of the plasma membrane, particularly under ruffles, and it is highly concentrated on the inner surface of the membrane near junctions connecting adjacent cells. Small amounts of p60”‘” were detected in the cytoplasm and in the perinuclear Golgi region of the cell. No significant localization was detected in control NRK cells or in NRK cells transformed by the Kirsten strain of murine sarcoma virus. The presence of ~60”” on the inner surface of the plasma membrane indicates that the changes in cell growth, cell shape and cell membrane structure noted in ASV-transformed cells are due to an initial action of p60”‘” at the cell membrane. Introduction The transformation of chick embryo fibroblasts and other cells by avian sarcoma virus (ASV) is accompanied by dramatic changes in cell shape and adhesion. These changes suggest that one site of action of the transforming gene product (src) is at the cell membrane (Pastan and Willingham, 1978). The product of the RSV src gene has been identified by immunological methods as a phosphoprotein with a subunit molecular weight of 60,000 (p60”“) (Collett and Erikson, 1978). This protein has been localized in ASV-transformed cells by immunofluorescence and found to be mainly in the cytoplasmic portion of the cell (Brugge, Steinbaugh and Erickson, 1978; Rohrschneider, 1979). We have recently developed a method that allows antigens to be located immunologically at the electron microscopic level (Willingham, Yamada and Pastan, 1978a, 1978b; Willingham and Yamada, 1979). This method has a number of advantages over fluorescence. First, individual organelles can be identified by their appearance at the high magnification and resolution produced by the electron microscope. Second, the fixation method used maintains the structure of all cellular organelles including

membranes. Third, the number of protein molecules detected by an antibody can be quantified. We have now used this method to determine the location of the ASV src gene product at the electron microscopic level. We find that p60”” is principally located at the inner surface of the plasma membrane. This location is in keeping with the rapid changes in cell shape and membrane ruffling that occur when the activity of the src gene is allowed to be expressed. Results Antisera An antiserum prepared in newborn rabbits as described by Brugge and Erikson (1977) was used to detect the location of ~60”” in Schmidt-Ruppin ASVtransformed NRK cells (SR-NRK). Before these experiments were performed, we tested the antiserum to see which proteins it precipitated from SR-NRK cells labeled with 35S-methionine. As shown in Figure 1 (lanes l-3) the major product detected has an M, of 60,000 (p60”“). A minor band with an M, of 76,000 (Pr76) was also detected when the autoradiograms were overexposed. This latter component is the precursor polyprotein coded for by the gag region (Vogt, Eisenman and Diggelman, 1975). These cells do not synthesize any envelope glycoprotein. Although the antibody titer against Pr76 was much lower than that against p60”‘“, we were able to make the antiserum monospecific for p60”‘” by neutralizing those antibodies reacting with Pr76. This was done by preincubating the immune serum with purified virus prior to immunoprecipitation of the labeled cell extract (Figure 1). The solutions used to lyse cells and virus (RIPA) contain Triton X-100, deoxycholate and SDS (see Experimental Procedures). RIPA could not be used as the lysis agent for viral absorption of the antisera in electron microscopic experiments because it disrupts membrane morphology. We therefore lysed the virus with saponin, the permeabilization agent used for electron microscopy. The saponin-lysed virus absorbed Pr76 selectively from the immune serum. The presence of saponin, however, caused a large increase in nonspecific precipitation, as shown in Figure 1 (lanes 4-6). We have overexposed these autoradiograms to show the Pr76 band in lane 5 (unabsorbed) and its absence from lane 6 (virus absorbed). The small band which migrates in the p60 region in lane 4 is a cell protein which is nonspecifically precipitated and, in fact, migrates slightly more slowly than ~60. Since immunoprecipitation in the presence of saponin results in nonspecific precipitation of cell proteins, these results are presented only to demonstrate that Pr76

Cdl 126

-76

r-60

Figure 1. lmmunoprecipitation of p60src and Pr76 from SWNRK Extracts by Immune Serum and Absorption of Antibodies against Pr76 by Viral Lysates Shown are autoradiograms of 7.5% polyacrylamide-SDS gels of 35Smethionine-labeled extracts of SWNRK cells immunoprecipitated as described in Experimental Procedures using immune serum (lanes 2 and 5), nonimmune serum (lanes 1 and 4) or immune serum absorbed with viral lysates (lanes 3 and 6). Absorption and immunoprecipitations were conducted using viral lysates made by incubating purified SR-ASV in RIPA buffer (lanes l-3) or in saponin (lanes 4-6).

can be absorbed out in the presence of saponin, and not to demonstrate the immunospecificity of the antisera. EGS Electron Microscopic Localization of ~60”’ to Plasma Membrane We have developed the EGS fixation-permeabilization procedure (Willingham et al., 1978a, 1978b; Willingham and Yamada, 1979) and combined it with the ferritin bridge localization method (Willingham, Spicer and Graber, 1971) to localize proteins in cells while maintaining excellent morphologic preservation. The ferritin cores shown in Figure 2 indicate the location of p60’“. There is a significant concentration of ~60’” on the inner aspect of the plasma membrane of SRNRK cells (Figures 2A-2C). Figures 2A and 2B show areas of plasma membrane having both smooth regions and surface protrusions such as ruffles. When

surface protrusions were examined, there was a somewhat higher concentration of ~60’” in ruffles than in smooth areas. There was also some ~60’” detected in the cytoplasm, but the concentration was much less than at the inner surface of the plasma membrane. When normal rabbit globulin or globulin from newborn rabbit (nonimmune) was tested, only a few cores were detected which were randomly distributed throughout the cytoplasm (Figures 2D and 2F). Figures 2E and 2F show examples of the perinuclear region of these cells, populated prominently by Golgi vesicles. These regions failed to show any significant concentration of ~60’” over that present in the rest of the cytoplasm. A striking finding is shown in Figure 3: we found intense labeling of gap junction regions where cells come in contact. NRK and Kirsten sarcoma virustransformed NRK (KNRK) also have these morphologic junctions at points of intercellular contact, but only SR-NRK showed localization to these structures with anti-p60’” (results not shown). As with other areas of the plasma membrane, ~60”” was located at the inner surface of the plasma membrane at these junctions. The gap junctions between SR-NRK cells were not labeled when normal rabbit globulin or nonimmune newborn rabbit globulin was used (Figure 3D). The ferritin bridge procedure involves a long sequence of globulins between the initial antigen and the ferritin cores (see Experimental Procedures). By measuring discrete structures such as 100 A filaments and microtubules using antibodies to their structural proteins, we have previously determined the length of the bridge sequence elements to be -250 A (our unpublished data). Thus we expected that antigens on the inner aspect of the membrane would show ferritin core locations 250 A away from the membrane. In addition, tangential sectioning of a convoluted membrane surface can add further apparent distance between a membrane antigen and ferritin cores. For these reasons, we considered ferritin located within 1000 A of the apparent membrane location to represent a membrane-related localization. To be certain that the localization observed was specific for p60”, the antiserum was absorbed with saponin-lysed SR-ASV, as described in Experimental Procedures. As shown in Figure 1, this absorption removes antibodies to Pr76 but not those to ~60”“. Localization with absorbed antiserum was identical to that with unabsorbed serum (results not shown). Thus the localization observed was not due to antibodies directed against viral structural proteins. When NRK and KNRK cells were tested with the antip60”” antiserum, no localization was observed (results not shown). Both NRK and KNRK cells have gap junctions, and those regions were also negative for ~60’“. We conclude that the localization seen in

RSV-Transforming

Protein

in Plasma

Membrane

127

Figure

2. Electron

Microscopic

lmmunocytochemical

Localization

of ~60””

to the Plasma

Membrane

of SWNRK

Cells

(A, B) Plasma membrane region with immune serum; (C) similar plasma membrane region using virus-absorbed serum; (D) plasma membrane region with nonimmune serum; (E) area near the nucleus with immune serum; (F) area near the nucleus with nonimmune serum. Arrows indicate the typical appearance of ferritin cores. (G) Golgi, (N) nucleus, (np) nuclear pore; lead citrate-bismuth subnitrate counterstain. Magnifications (AD) 38.000X; (E, F) 30,762X.

SR-NRK cells represents We have considered tration of label at the

the location of ~60”“. the possibility that the concenplasma membrane is due to

a

permeability gradient characteristic of the EGS permeabilization-fixation procedure. We are convinced, however, that this is not the case, since numerous

Cell 126

Figure

3. Electron

Microscopic

lmmunocytochemical

Localization

of ~60””

to the Plasma

Membrane

in Gap Junction

Regions

in SR-NRK

Cells

(A), (B) and (C) show localization of ferritin cores (arrowheads) to the plasma membrane in gap junctions between adjacent SR-NRK cells. (C) is from an experiment using virus-absorbed immune serum. (D) shows a similar region in cells incubated with nonimmune serum (arrows show ends of the gap junction). L = lipid. (A, C. D) lead citrate-bismuth subnitrate counterstain: (B) uranyl acetate-lead-bismuth counterstain. Magnification 69,666x.

other antigens which are present in mic locations have been localized dure. These include tubulin (M. C. Yamada and I. Pastan, manuscript 100 A filament protein (F. Cabral,

deeper cytoplasusing this proceWillingham, S. S. in preparation), M. C. Willingham

and M. M. Gottesman, macroglobulin (M. S. I. Pastan, manuscript (S. S. Yamada, M. C. manuscript in preparation).

manuscript in preparation), (YeWillingham, S. S. Yamada and in preparation) and fibronectin Willingham and K. M. Yamada,

RSV-Transforming 129

Table

Protein

1. Morphometric

in Plasma

Quantitation

Membrane

of Localization

Antibody

Localization

of ~60”” (Ferritin

in SR-NRK Cores

Cells

per $)

Plasma Membrane (
Plasma Membrane Gap Junctions (<250 A)

Total Cores Counted

5030

3156

Antiserum

Free Cytosol

Golgi (Perinuclear) Region

Immune

287

256

853

98

97

141

Nonimmune

74

605

Assuming a spherical cell diameter of 16 p. with 50% of the cell volume occupied by nucleus and 50% of the remaining cytoplasmic volume occupied by cytoplasmic organelles, the free cytosol volume in such a cell would be -525 $. The surface area of such a spherical cell would be -800 p* and, considering a 0.1 rr (1000 A) region near this membrane, the volume of the plasma membrane region would be -80 p3. Together with the data shown in this table, -60% of ~60”” in the cell would then be associated with the cytosol and -40% would be within 1000 A of the plasma membrane. -

Quantitation of Distribution of ~60”” Although visual examination of the electron micrographs provides convincing evidence of the concentration of p60”” to the plasma membrane, the ferritin label lends itself to morphometric quantitation. In this procedure, the number of ferritin cores in sections 800 A thick were counted and the data were expressed as concentration per $. Table 1 shows quantitation of the distribution of p60”” in cytosol, plasma membrane, Golgi and gap junctions in SR-NRK cells. The differences in concentration are also shown in Figure 4, which emphasizes the concentration of p60 src on the inner aspect of the plasma membrane (it is particularly concentrated at locations where the plasma membrane is part of a gap junction). Since the intercellular junctional complex structure in these cells may not be morphologically identical to similar structures in other cell types (Revel and Karnovsky, 1967; Gilula, 1977) we have examined it using conventional fixation and processing methods to characterize its appearance further, since we thought it possible that the EGS procedure might alter the morphology of these structures. Figure 5 shows the morphologic appearance of these junctional complexes in SR-NRK cells fixed, processed and counterstained by conventional methods. In these junctions, there is a uniform space between the membranes of adjacent cells containing a particulate proteinaceous material. Because the bilayer structure of the plasma membrane in these cultured cells has been difficult to preserve, the appearance of these junctions probably reflects the lack of bilayer preservation. Otherwise, these structures are similar to the gap junctions decribed in other cell types (Gilula, 1977). lmmunofluorescence Brugge et al. (1978) and Rohrschneidger (1979) have reported that ~60”” is principally found in a cytoplasmic location. We have also conducted immunofluorescence experiments with acetone-fixed cells (Figure 6) and we observed, as did Rohrschneider (1979) that most of the apparent fluorescence localization is in the cytoplasm in the perinuclear region and occasionally at points of contact between adjacent cells (Figures 6C, 6D and 6E). We also observed some

Cytosol

Figure 4. Quantitation Morphometric Analysis

of p60’”

Plasma

Membrane Localization

by Electron

Plasma Membrane

Junctions Microscopic

The concentration of label was determined as described in Experimental Procedures. These “specific” values represent the difference between the concentration of label with immune versus nonimmune sera, as shown in Table 1.

fluorescence at the periphery of individual cells (Figures 6C and 6E). Fluorescence localization using acetone or formaldehyde-acetone fixations suffers from the lack of resolution inherent with light microscopy, as well as the severe alteration of subcellular morphology previously noted with the fixation procedures used for fluorescence localization (Willingham and Yamada, 1979). An example of the destruction of the morphology of intracellular organelles is presented in Figure 7A. The cell shown was first acetone-fixed, then post-fixed in glutaraldehyde and osmium, and embedded for electron microscopy. It is apparent that none of the intracellular cytoplasmic structures are recognizable. Interpretation of intracellular detail from fluorescence results with acetone-fixed cells must therefore be undertaken with caution. A further problem with the fluorescent microscopic approach is that entire cells are viewed from above in two dimensions. Cultured cells are not symmetrical.

Cell 130

Figure

5. Morphologic

Appearance

of Gap Junctions

in SR-NRK

Cells Processed

by Routine

Fixation

and Embedding

Procedures

These micrographs demonstrate the appearance of gap junctions (between the arrows) in SR-NRK cells fixed in glutaraldehyde and osmium in a routine fashion as described in Experimental Procedures. (A) Ethanol dehydration: (B) ethanol-propylene oxide dehydration. Uranyl acetate-lead citrate counterstain. Magnifications (A) 57,905~; (B) 93,190x.

They are considerably thicker in the perinuclear region than at the cell periphery. If, therefore, a fluorescent molecule is uniformly distributed throughout the cytoplasm, the perinuclear region will be brightest because it is thickest and has more fluorescent molecules. Thus we cannot interpret the images in Figure 6 to label in the perreflect a concentration of fluorescent inuclear region, since that region is much thicker than the projection of the thinner periphery of the cell. Furthermore, the cell-to-cell contact points are not easily related to known subcellular structures without detailed electron microscopic information about the morphology of these regions. When one views these cells by phase microscopy after they have been acetone-fixed and embedded for electron microscopy, the perinuclear region shows a considerable concentration of density (Figure 7B) similar to the appearance of the fluorescent perinuclear regions seen in Figure 6. This density probably derives from the tendency for organelles to concentrate in the perinuclear region in this cell type (Figures 2E and 2F), along with the considerably increased cell thickness in the nuclear region. In some of the cells in Figure 6, there is concentration of fluorescence at the rounded edge of cells. Although this fluorescence could reflect some concentration at the cell membrane, it required localization at the electron microscopic level to establish this in a convincing manner.

Discussion In this paper, we have established that the product of the transforming gene of ASV p60”” is concentrated at the inner surface of the plasma membrane of SRNRK cells. Although this protein is present in very small amounts in transformed cells, immunological localization at the electron microscopic level is a very sensitive technique and readily detects ~60”“. Significance of Plasma Membrane Localization In addition to inducing abnormal cell growth, ~60”” is responsible for dramatic changes in cell shape, plasma membrane structure (ruffles, blebs and microvilli) and plasma membrane function (adhesion to substratum, fall in adenylate cyclase activity and altered transport of small molecules) (Pastan and Willingham, 1978). The earliest changes observed when cells infected with temperature-sensitive transformation mutants of ASV (such as tsNY68) are shifted to the permissive temperature are formation of surface ruffles, cell rounding and loss of adhesion. Our finding that ~60”” is present at the inner surface of the plasma membrane suggests that these early rapid changes are due to a direct action of the transforming protein on the cell membrane. Indeed, the increased concentration of ~60”” in membrane extrusions such as ruffles suggests that these are formed at sites where There has been much specu~60 *” is concentrated. lation that cell shape is controlled by the binding of

RSV-Transforming 131

Figure

Protein

6. lmmunofluorescence

in Plasma

Membrane

Appearance

of SR-NRK

Cells Fixed with Acetone

Cells were fixed in acetone and incubated as described in Experimental Procedures. Normal rabbit globulin (A) or nonimmune newborn rabbit serum (B) show very little fluorescence localization: (C), (0) and (E) show the pattern using immune serum to p60”“. Besides an apparent perinuclear concentration of label, these cells show bright fluorescence at the occasional intercellular connection (arrowheads), but also an apparent concentration of fluorescence at the free cell edge (arrows). Rhodamine epifluorescence. Magnification 530x.

structural membrane, (McClain,

proteins such as actin and that ~60’” might Maness and Edelman,

or myosin alter this 1978).

to the cell association The mor-

phology of cells fixed by the EGS procedure is excellent, and allows 60 A filaments to be visualized and even localized by antibodies to actin (Willingham et

Cell 132

Figure

7. Morphologic

Appearance

of SR-NRK

Cells Fixed

with Acetone

Cells fixed using the procedures described in Figure 6 were processed for electron microscopy with post-fixation in glutaraldehyde and osmium. Ultrastructural preservation is minimal, as shown in (A), with a concentration of unrecognizable organelle remnants in the perinuclear region (arrows). By phase-contrast microscopy (6) in the intact block, these cells showed a concentration of density in the perinuclear regions (arrows). Magnifications (A) 7000X; (8) 1360X.

al., 1978b; also unpublished observations). While we have not detected any association of ~60”‘” with 60 A filaments or other structural proteins, it remains possible that the presence of ~60”” at the inner surface of the plasma membrane directly blocks the association of cytoskeletal elements with membrane proteins. In trying to rationalize how a single transforming protein such as ~60”” could produce the dramatic changes in cell membrane structure and function previously described, as well as alterations in the synthesis of collagen, fibronectin and hyaluronic acid, we pointed out how the actions of transforming proteins resemble the actions of peptide hormones such as insulin and epidermal growth factor (Pastan and Willingham, 1978), and suggested that the plasma membrane is the logical site of action of transforming proteins. This suggestion has now been confirmed. However, peptide hormones bind to the outside of the plasma membrane, whereas ~60”” is bound to the inner surface. Although insulin and EGF enter cells, the ability of these hormones to enhance amino acid transport and growth does not require their entry into cells (A. LeCam, F. R. Maxfield, M. C. Willingham and I. Pastan, manuscript in preparation; F. R. Maxfield, P. J. A. Davies, L. Klempner, M. C. Willingham and I. Pastan, manuscript in preparation). We speculate that growth-promoting peptide hormones and ~60’” inter-

act with similar membrane proteins that promote cell growth and the metabolic alterations related to growth. We believe that the concentration of ~60”” is the important factor in determining its major site of action. From the data shown in Table 1, however, it can be seen that of the total amount of ~60”” in the cell, 60% is in the cytosol. Thus it is also possible that some or all of the action of ~60”” might occur at sites in the cytosol which have no morphologic distinction by these methods. ~60”” in Junctional Complexes At present we have no clear idea why p60”” accumulates at junctional complexes. Not all cells capable of being transformed by ASV have these complexes, and ASV does not induce their formation. It is possible that the junctional complexes have a role in contact inhibition of growth of NRK cells and that p60sfc prevents them from carrying out this function. It is probin these regions beable that ~60”‘” IS concentrated cause it binds to one of the components of the complex. ~60”” may represent a significant fraction of the total protein of the complexes, and isolation of the complexes may assist in the preparation of pure ~60’” Problems with Fluorescence Localization lmmunofluorescence localization in cultured

cells

is

RSV-Transforming 133

Protein

in Plasma

Membrane

usually accompanied by two problems: first, the fixation procedures commonly used do not preserve cellular organelles very well and drastically alter some morphologic structures, and second, the visualization of localization is made by looking at a two-dimensional image of a three-dimensional cell. These problems are not severe when localizing discrete, widely separated structures such as vesicles or microtubules at peripheral locations in flattened cells. Diffuse localization patterns, however, create particularly difficult interpretive problems. The perinuclear region in most cells, including SR-NRK, is much thicker than more peripheral parts of the cell, and small amounts of diffuse label look as if the antigen is highly concentrated because of this. Another problem with the fluorescent results was that cytoplasmic perinuclear localization was not seen in all the cells; many cells were negative. There was no variation in the localization of ~60”” to the plasma membrane in the electron microscopic studies. Finally, our inability to detect ~60”” in the Golgi was not due to technical problems associated with the EGS procedure, since we have successfully localized a,-macroglobulin and fibronectin to the inside of the Golgi (Willingham et al., 1978a; S. S. Yamada, M. C. Willingham and K. M. Yamada, manuscript in preparation) and clathrin to the area around the Golgi (J. Keen, M. C. Willingham and I. Pastan, manuscript in preparation) with this procedure. Other Transforming Proteins The high resolution of the electron microscopic method used here has allowed us to localize a trace antigen in SR-NRK ceils. The method can be used to determine the precise intracellular location of other transforming proteins to which specific antisera are available. Studies of this sort are now in progress. Experimental

Procedures

Cells and Viruses Control NRK and NRK transformed by SR-ASV (SR-NRK) or KiSV (KNRK). provided by E. Scolnick. were grown in Dulbecco’s minimal essential medium supplemented with 10% calf serum. Primary cultures of chick embryo fibroblasts (CEF) were prepared from 10 day old embryonated eggs. infected with cloned SR-ASV subgroup D and grown in Ham’s FlO medium supplemented with 10% tryptose phosphate broth, 0.5% beef embryo extract and 5% calf serum. SR-ASV were purified from the supernatants of secondary SR-CEF by banding twice on sucrose gradients. Antiserum Immune serum was prepared by injecting newborn rabbits with -10’ ffu of purified SR-ASV, as described by Brugge and Erikson (1977). Pooled bleedings from one rabbit were used for these experiments. Immune serum was made monospecific for ~60”” by incubation with purified SR-ASV. 10 PQ per 5 pl serum, either in RIPA buffer or in TBS containing 0.05% saponin. lmmunoprecipitation and Virus Absorbtion of Antiserum Extracts were prepared from cells that were labeled for 2 hr at 37°C with 200 pCi per 100 mm dish of ?S-methionine. The cells were then washed twice with Earle’s balanced salt solution and twice with Tris-

buffered saline (pH 7.2) containing 1 mM EDTA, lysed in 1 mM RIPA buffer [TBS (pH 7.2), 1% Triton X-100, 1% deoxycholate, 0.1% SDS]. The extracts were clarified by centrifugation at 100,000 X Q for 30 min. To 5 ~1 of antiserum, we added 5 pl of virus in the presence of 0.2 ml of RIPA or 0.2 ml 0.5% saponin, and incubated the mixture for 30 min at 1 “C. As a control, the antiserum was incubated with RIPA or saponin without virus. The suspensions were then centrifuged at 100,000 x g for 60 min to remove any unlysed virus particles. To all supernatants, we added 0.3 ml RIPA and 0.3 ml clarified cell extract. These were incubated for 30 min at 1 “C before the addition of 100 ~1 of boiled S. aureus, and the incubation continued for another 30 min. The immunoprecipitates were washed 3 times in 1 ml RIPA containing 2.5 M potassium chloride, and then once in RIPA and once in 10 mM Tris-HCI (pH 7.2) containing 0.5% NP40. The final immunoprecipitate pellets were taken up in 60 ~1 SDS gel mix [70 mM TrisHCI (pH 6.8), 12% glycerol, 3% SDS, 0.01% bromophenol blue, 10% mercaptoethanol] and boiled for 5 min to dissociate the antigenantibody complex from the S. aureus. The samples were then centrifuged, and the supernatants were analyzed on 7.5% polyacrylamide SDS gels (Laemmli. 1970). The gels were fixed and stained in a solution of 50% TCA-0.25% Coomassie blue, destained, fluorographed with DMSO and PPO (Laskey and Mills, 1975), and dried. Autoradiograms were prepared by exposing the dried gels to Kodak X-Omat film at -70°C for 24-48 hr. lmmunofluorescence Cells were fixed in acetone and processed by a method similar to that described by Rohrschneider (1979). Since cells were grown in 10 cm2 plastic dishes, 80% acetone/H20 was used as the primary fixative either with or without air drying. After fixation and washing in PBS, the cells were incubated in a 1:20 dilution of rabbit anti-p60”” immune serum, normal rabbit globulin or nonimmune newborn rabbit serum, for 60 min at 37°C in PBS to which 2 mg/ml bovine serum albumin and 2 mg/ml normal goat globulin (BSA-NGG-PBS) had been added. All globulin solutions were filtered through 0.22 @ Millipore filters. Unlike Rohrschneider. we did not “cleanse” our fluorescent indirect conjugate by in vivo absorption. Rather, goat anti-rabbit IgG conjugated with rhodamine (Cappel Labs) was preabsorbed in the presence of BSA-NGG-PBS to dishes of acetone-fixed SR-NRK cells. Specifically, 75 yl of the goat anti-rabbit rhodamine conjugate (-12 mg/ml) were diluted to 3 ml in BSA-NGG-PBS and serially absorbed on six 100 mm (50 cm*) dishes of acetone-fixed confluent SR-NRK cells for 10 min each at 37°C. The supernatant from these absorptions was Millipore-filtered and used in a final volume of 3 ml in BSA-NGGPBS as the second step for fluorescence labeling. Failure to absorb the indirect conjugate in this manner resulted in considerable intracellular background which obscured specific localization. The absorbed conjugate (second step) was incubated with the cells for 30 min at 37°C following a wash after the first step in BSANGG-PBS. After this second incubation step, the cells were washed in BSA-NGG-PBS (three times) and then in PBS (three times), and finally mounted in glycerol buffer medium under a circular coverslip. Fluorescence was observed using a Zeiss RA microscope equipped with rhodamine epifluorescence illumination and photographed on Polaroid 107 (ASA 3000) film using a 40X. (N.A. 1.0) oil immersion lens (exposure time 90 set). Electron Microscopic Localization Cells were fixed and permeabilized using the EGS procedure (WilIinQham, et al., 197Sa, 1978b). Briefly, the primary fixative was assembled by initially preparing a stock buffer solution by mixing 100 mM Na-phosphate solution in equal parts with Dulbecco’s (Ca”-, Mg+‘-free) PBS. To this buffer solution we added Tris base to a concentration of 1.4% and adjusted the pH with HCI to 7.0. This buffer solution therefore contained 50 mM POn. 50% concentrated PBS and 1.4% Tris (pH 7.0). TO this buffer at 23°C we simultaneously added 50% Qlutaraldehyde (Tousimis) to a final concentration of 0.2% and powdered EDC (ethyldimethyl aminopropyl carbodiimide) (Sigma) to a final concentration of 1%. At this point, a timer was started. At 3 min. the pH was

Cell 134

adjusted to 7.0 with 1 N NaOH (it had fallen to pH 6.6). At precise/y 4 min, the fixative solution was added to 10 cm2 dishes of cells that had previously been washed at 23’C with PBS. This fixative solution was left on the cells at 23’C for 7 min and then washed off with PBS. Membrane permeabilization was then achieved by incubating the fixed cells with 0.05% saponin (Sigma) in PBS, 4 mg/ml normal goat globulin and 2 mM EGTA for 30 min at 23°C. This saponin-globulin-EGTA-PBS solution was used for all subsequent antibody incubations and washes. Each antibody incubation was for 30 min at 37’C. We used the ferritin bridge procedure (Willingham et al., 1971) for immunocytochemical labeling. The sequential incubations were first, rabbit anti-p60s” serum normal rabbit serum or nonimmune newborn rabbit serum at I:20 dilution; second, goat anti-rabbit globulin at 1: 20 dilution: third, rabbit anti-ferritin (Cappel) which had been affinitypurified against ferritin (100 pglml); and fourth, ferritin (100 pg/ml) (Sigma). At the end of these incubations, the cells were washed in PBS and post-fixed in 3% glutaraldehyde in PBS at 23’C for 10 min, followed by 1.5% 0~0, in PBS for 10 min at 23°C. This was followed by dehydration in 70-1009/o ethanol and immediate immersion in Epon 812 polymerization mixture at 23°C for 3 hr. Fresh Epon mixture was added and the dishes were incubated at 58’C for 48 hr. The dishes were mechanically separated from the epoxy embedments, and small blocks were cut using a jeweler’s saw and mounted in a vise chuck. Thin sections were cut using a Sorvall MTP-B ultramicrotome and a diamond knife, and mounted on 200 mesh uncoated copper grids. Post-sectioning staining was either with 1% uranyl acetate followed by lead citrate and bismuth subnitrate, or by lead citrate-bismuth subnitrate alone. For routine morphology (as in Figure 5). dishes of cells were fixed in 3% glutaraldehyde in PBS for 10 min at 23°C. followed by 1.5% OsOa in PBS for 10 min at 23°C. Cells were dehydrated in ethanol and either embedded in situ in Epon 812 (as described above) or gently detached by shaking, dehydrated in propylene oxide and embedded as a pellet after centrifugation at 15,000 x g. Sections from those blocks were then counterstained with uranyl acetate saturated in methanol followed by lead citrate. Sections were viewed with a Hitachi HU-12A electron microscope at 50 kV using a 30 p objective aperture. Quantitative lmmunolocalization For quantitation of ferritin localization, groups of randomly selected cells were counterstained with lead citrate-bismuth subnitrate, photographed at 24,000X and printed at a final magnification of 60,000x. Fields from free cytosol, Golgi regions, plasma membrane regions and gap junctions were chosen at random from cells incubated with either nonimmune newborn rabbit serum or rabbit anti~60’” serum. From the prints, areas of free cytosol were selected and outlined. Similarly, segments of plasma membrane and a 1000 A wide adjacent segment of cytosol (to allow for tangential sectioning) were outlined. Areas of gap junctions and adjacent 250 A wide areas of cytosol ware chosen and outlined. Each of these outlined areas was cut out from the prints with scissors, along with premeasured standard regions of known area. The paper was weighed and the area (in $) was calculated for each field represented. Ferritin cores were easily discernible, and were counted and tallied for each segment. The areas of the fields represented in each morphologic group were totaled, along with their tally of ferritin cores. By assuming an average section thickness of 800 A. we could calculate the volumes represented by these fields. After calculating both total volume and total number of ferritin cores for each morphologic group, we obtained a concentration of ferritin cores per p3. The level of nonspecific background in each-of the morphologic regions was quite similar for both the normal serum and the nonimmune newborn rabbit serum (-100 ferritin cores per p3). The large number of cores counted (~3500) provided errors of c5% for the categories shown in Table 1 and Figure 4.

Acknowledgments The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact. Received

April 19, 1979;

revised

June 12. 1979

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