3T3 and SV3T3 mouse embryo cells and a variety of other monolayer cell lines can be induced to form and shed plasma membrane vesicles by exposure to sulphydryl blocking agents including formaldehyde and N-ethyl malemide. Morphological studies show that multiple vesicles are formed and released from individual cells and that the vesicle membrane is continuous with the plasma membrane of the cell. Vesicles measure from 0 ·1 to 15 μm in diameter and are free of detectable contamination with cytoplasmic membranes and organelles. Vesicles also show a 10-fold enrichment in the plasma membrane marker enzyme 5’nucleotidase and are devoid of detectable NADH-cytochrome C reductase and succinic dehydrogenase activity which are marker enzymes for endoplasmic reticulum and mitochondria, respectively. Vesicles have a high cholesterol: phospholipid ratio and show enrichment in sphingomyelin content. They contain receptors for Con A and WGA, approximately 20 size class polypeptides and intra-membranous particles. These results suggest that vesicles are derived from and have the general characteristics of plasma membranes.

The cell surface of normal and transformed cells has been studied in detail. Differences in lectin agglutinability (Burger & Marten, 1972), in cell surface morphology (Porter, Todaro & Fonte, 1973), in the rate of nutrient transport (Isselbacher, 1972), in adenylate cyclase activity (Peery, Johnson & Pastan, 1971) and in the composition of glycoproteins and glycolipids (Hynes, 1976) have been reported and it has been suggested that there are differences in the interaction of the plasma membrane and the microskeleton (Edelman, 1976). These data for the most part have been obtained from studies using intact whole cells, whole cell homogenates or relatively crude microsomal-membrane fractions. Very few analytical studies have been performed on highly purified plasma membranes of cells grown in tissue culture. An important reason for this shortcoming is the difficulty in isolating purified plasma membranes.

In this paper we describe in detail a new and novel approach for the isolation of plasma membrane vesicles from monolayer tissue culture cell lines, in particular 3T3 and SV3T3 mouse embryo cells. The method is based on the observation that a variety

All correspondence to be sent to: Robert E. Scott, M.D., at the above address. of sulphydryl blocking reagents induce the formation and release of vesicles from the plasma membrane (Scott, 1976). Vesicles released into the medium are free of contamination with cytoplasmic membranes but do contain soluble cytosol components which can be easily removed by lysis and washing of the vesicles to yield preparations of pure plasma membrane. Such specimens provide a new and valuable source of material to study the compositional and structural characteristics of the plasma membrane.

Cell lines

The cell lines used in this study included Balb/c 3T3 mouse embryo cell (clone A31), methyl-cholanthrene (MCA) and Simian Virus 40-transformed Balb/c 3T3 mouse embryo cells (a gift of Dr George Todaro). These cells were grown in Dulbecco’s minimal essential medium containing 10% calf serum (Grand Island Biological Co., Grand Island, N.Y.) at 37 °C in a humidified atmosphere containing 10 % CO2 in air. Antibiotics were not added to the medium and no evidence of fungal or bacteriological contamination was observed in cells used in these studies. These cells were also tested and were found to be negative for mycoplasma contamination by electron microscopy and cultural analysis. The saturation density for 3T3 cells used in these studies was approximately 4 × 106 cells/cm3 and for SV3T3 cells 2 × 105 cells/cm*.

Selected experiments were also performed on other cell lines detailed in Table 2, p. 233. Swiss 3T3 cells were kindly provided by Dr Jack Sheppard; AKR cells, methylcholanthrene-transformed AKR cells and XC cells by Dr Harold Moses; Lβ myoblasts by Dr David Shubert; neuroblastoma cells (N1300) by Dr Elliot Richelson and owl monkey kidney cells by Dr Gary Pearson. Human leukemic monocytes (J-111) were purchased from the American Type Tissue Culture Collection (ATCC). Guinea-pig peritoneal exudate macrophages were produced by sterile peritoneal irritation with Marcol 50 oil. Secondary cultures of low passage human skin fibroblasts were also used.

Table 2.

Effect of a plasma membrane vesiculant on different cell populations

Effect of a plasma membrane vesiculant on different cell populations
Effect of a plasma membrane vesiculant on different cell populations

Plasma membrane vesicles

Plasma membrane vesiculation was tested by a standard method. Monolayer cell cultures were grown to approximately 70% confluence in 60-mm Petri dishes (Falcon; Oxnard, CA) or on glass coverslips. Cells were washed 3 times in isotonic phosphate-buffered saline (pH 7·4) containing 0·75 mM CaCl2 and 0·5 mM MgCl2 (CMPBS). Cells grown in suspension were sedimented by centrifugation at 100 g for 15 min and then washed 3 times in CMPBS. Cells were then incubated in specific plasma membrane vesiculants. Vesiculation was scored on a scale of zero to four.

Bulk isolation of plasma membrane vesicles employed incubations of cells for 2 h at 37 °C in a vesiculant of 25 mM f0rmaldehyde-2 mM dithiothreitol (DTT) prepared in phosphate-buffered saline containing 0·75 mM calcium and 0·5 mM magnesium (CMPBS). Vesicles were decanted from the cells and passed through a glass wool column to remove detached cell aggregates. This solution was sedimented by centrifugation at 30000 g for 30 min at 4 °C. The resulting translucent pellet was gently resuspended so as to leave any residual debris as a small dense white pellet at the bottom of the tube. Vesicles were washed 3 times in 50 mM Trisbuffered saline, pH 7 · 4, and finally sedimented at 30000 g for 30 min at 4 °C. This plasma membrane pellet was used in the studies presented in this paper, unless otherwise stated.

Quantitation of the amount of membrane protein was performed both by Lowry, Rosebrough Farr & Randall (1951) and fluorescamine (Bohlen, Stein, Dairman & Udenfriend, 1973) procedures. Bovine serum albumin (BSA) was employed as the standard. Comparable results were obtained with both assays.

Enzymatic characterization

Enzymes, which were assayed according to published procedures, included 5’nucleotidase (Michell & Hawthorne, 1965), NADH-cytochrome C reductase (Hatefi & Rieske, 1967) and succinic dehydrogenase (Khoww & McCurdy, 1969). Enzyme assays were performed on plasma membrane vesicles, on whole cell homogenates and on homogenates of whole cells exposed in situ to 25 mM formaldehyde-2 mM DTT-CMPBS for 30 min at 37 °C and then washed.

The virtual abεence of DNA in vesicles was demonstrated by the diphenylamine procedure (Burton, 1956) and by the demonstration that no detectable quantity of [3H]thymidine was present in TCA-precipitated membranes isolated from cells pulsed for 16 h in [3H]thymidine (50 Ci/mmol, New England Nuclear, Boston, M A) to label DNA.

Morphological studies

Specimens of cells shedding vesicles and of isolated vesicles were fixed in 2·5 % glutaraldehyde —0 ·1 M cacodylate buffer (pH 7 · 4) at room temperature for 1 h. They were then washed 3 times in 0 ·1 M phosphate buffer (pH 7 · 4) and postfixed in 2 % osmium tetroxide for 30 min at room temperature. In some experiments specimens were prefixed in 2 % calcium permanganate for 30 min at room temperature or fixed in glutaraldehyde containing 8 % tannic acid.

The latter 2 treatments increased the yield of vesicles attached to cells but did cause loss of cytoplasmic detail. Fixed specimens were dehydrated and embedded in Epon. Sections were stained with uranyl acetate and lead citrate with or without en bloc staining with aqueous uranyl acetate. Thin sections were examined with a Philips EM 201 electron microscope.

Specimens for scanning electron microscopy were routinely fixed in 2 · 5 % glutaraldehyde —0 ·1 M phosphate buffer (pH 7·4) for 1 h at room temperature and then for 12 h at 4 °C. Some specimens as described above were prefixed in 2 % calcium permanganate prior to glutaraldehyde treatment. Fixed specimens were washed in 0 ·1 M phosphate (pH 7 · 4), dehydrated and critical-point-dried in a Sorvall apparatus (Sorvall Istruments, Newtown, CT.) with Freon 13. They were then coated with gold-palladium in a Denton vacuum evaporator and examined with an ETEC stereoscan electron microscope operating at 20 kV.

Polyacrylamide gel electrophoresis

Purified plasma membranes were prepared from vesicle suspensions by addition of 50 mosmol buffer followed by lysis by nitrogen cavitation (1·725 × 103 kN m−2 for 5 min). The membranes were then collected by sedimentation at 250000 g for 1 h. Thereafter the membranes were washed once and resedimented. This procedure releases soluble cytoplasmic proteins trapped within the vesicles. Purified plasma membrane pellets were solubilized in a solution containing 1 % SDS-10 mM DTP in 0 ·01 M PO4 buffer at pH 7·4. This suspension was heated in a boiling water bath for 5 min. Urea was added to a final concentration of 4 to 6 M and this solution was then sedimented at 20000 g for 15 min at room temperature to remove debris and any insoluble material that was present. Known quantities of protein (25 to 75 μg) were added to 5 % acrylamide gels containing crosslinking agents at a concentration previously reported (Segrest & Jackson, 1972). Polyacrylamide gel electrophoresis was performed by the method of Fairbanks, Steck & Wallach (1971). Coomassie blue-stained gels were scanned with a Beckman Acta II spectrophotometer equipped with a linear transport device. The molecular weights of individual polypeptides were calibrated using β galactosidase, bovine serum albumin and cytochrome C as standards.

Lectin-induced vesicle agglutination

The agglutinability of plasma membrane vesicles derived from 3T3 and SV3T3 cells with Concanavalin A (Con A) and wheat germ agglutinin (WGA) was tested. Con A was purchased from Sigma Co., St Louis, MO and WGA was purchased from Miles-Yeda LTD, Elkart, IN. Washed 3T3 and SV3T3 plasma membrane vesicles were resuspended in phosphate buffered saline containing lectins. Incubations were carried out at 37 °C for 30 min. Vesicles were then sedimented by centrifugation at 3 0 000 g for 30 min at room temperature and gently resuspended. The degree of vesicle agglutination for control and lectin-exposed samples was determined by phase microscopy. Vesicle agglutination was scored from 0 to + + + +. Agglutination of + + + + was scored when > 75 % of the vesicles agglutinated; + + +, >, 50 %; + + >, 25 %, and +, < 25 %.

Phospholipid and cholesterol analysis

The lipid composition of 3T3 and SV3T3 whole cell homogenates and 3T3 and SV3T3 plasma membrane vesicles was determined by extraction of aqueous membrane pellets with cold chloroform: methanol (2:1) according to the procedure described by Perkins & Kummerow (1976). Free cholesterol and total phospholipid were determined using modified versions of the procedures reported by Searcy & Berquist (1960) and Eng & Noble (1968), respectively.

Reagents

All reagents were purchased from commercial sources in the highest grade available. Formaldehyde was prepared from paraformaldehyde (Aldrich Chemical Co., Milwaukee, WI) by dissolving 50 g in 300 ml H2O. This solution was heated at 60 °C for 1 h then NaOH (1 M) was added dropwise until the turbid solution cleared. This 10 % formaldehyde solution was prepared at weekly intervals and was stored at 4 °C under nitrogen after filtration through What man no. 1 filter paper.

Induction of plasma membrane vesiculation

A variety of sulphydryl blocking agents (Carter, Fox & Kennedy, 1968; Hayat, 1970; Hochster, Kates, Quastel & Glick, 1972; Lewin, 1956; Means & Feeney, 1971; Skov & Hilberg, 1965; Webb, 1966 a, b) induce plasma membrane vesiculation in cultured mammalian cells (Table 1). Formaldehyde, and N-ethyl malemide were in general the most effective vesiculants. No observable vesiculation could be detected with alcohols, acids, ketones, or with succinimide, a maleimide derivative which does not bind to free sulphydryl groups (Webb, 1966 a, b). Reagents which only reduce disulphide bonds, such as dithiothreitol, 2-mercaptoethanol, and L-cysteine, also did not induce vesiculation. Table 2 shows that the phenomenon of plasma membrane vesiculation was observed in a wide variety of monolayer cell cultures. These include fibroblasts, myoblasts, macrophages and cells of neural origin. Aldehyde-induced vesiculation was observed in cell lines of mouse, rat, guinea pig, monkey and human origin; in primary and secondary cultures and in established cell lines. These various cells all grew as monolayer cultures. The plasma membrane vesiculation technique is not effective for suspension cultures, at least under the conditions used in these experiments.

Table 1.

Effective plasma membrane vesiculants in 3T3 cells

Effective plasma membrane vesiculants in 3T3 cells
Effective plasma membrane vesiculants in 3T3 cells

Morphological characteristics of vesiculation in 3T 3 and SV3T3 cells

Fig. 1 illustrates plasma membrane vesiculation in 3T 3 and SV3T3 cells. Untreated 3T3 cells show a polygonal cell shape with little cell overlapping and no evidence of vesiculation (Fig. IA). Following exposure to formaldehyde for 15 min, minute optically dense spherical protrusions appear on the cell surface. These small blebs enlarge and after incubation for 30 min numerous 0· 1 - to 1· 0-μm projections can be seen. The formation and release of vesicles progresses rapidly at 37 ° C and after 60 to 90 min large quantities of cell surface vesicles can be seen in the medium (Fig. 1 B).

Fig. 1.

Phase-contrast micrographs of 3T3 (A, B) and SV3T3 (c, D) mouse embryo cells: before (A, C) and after (B, D) the formation and shedding of plasma membrane vesicles, A, × 195; B, ×205; c, ×210; D, × 195.

Fig. 1.

Phase-contrast micrographs of 3T3 (A, B) and SV3T3 (c, D) mouse embryo cells: before (A, C) and after (B, D) the formation and shedding of plasma membrane vesicles, A, × 195; B, ×205; c, ×210; D, × 195.

The process of vesiculation continues for approximately 2 h. Fig. 1c and D compare the appearance of native SV3T 3 cells and those shedding vesicles. The morphological appearance of cells incubated in other vesiculants was comparable.

Fig. 2 illustrates plasma membrane vesicle formation in 3T 3 and SV3T 3 cells by scanning electron microscopy. Vesicles initially develop at the cell margin and appear as small focal swellings (Fig. 2A, B). Subsequently, vesicles are formed and released from different regions of the cell surface. Up to ten plasma membrane vesicles have been observed to arise from an individual cell. In many cases vesicles of different sizes and of different sites of origin can be observed on an individual cell (Fig. 2B) and in some cases small projections are observed on the vesicle itself (Fig. 2c). These small projections are also observed in negatively stained preparations of isolated vesicles.

Fig. 2.

Scanning electron micrographs of 3T3 (A) and SV3T3 cells (B, C) forming plasma membrane vesicles. A, × 1760; B, × 2400; C, × 1560.

Fig. 2.

Scanning electron micrographs of 3T3 (A) and SV3T3 cells (B, C) forming plasma membrane vesicles. A, × 1760; B, × 2400; C, × 1560.

By transmission electron microscopy it is apparent that vesicles develop as saccular outpouching of the plasma membrane (Fig. 3). The vesicles show a unit membrane which is continuous with the plasma membrane of the cell. The cytoplasmic surface of the plasma membrane vesicle contains adherent fibrous material and cytosol but no organelles and very few polysomes. Ultrastructural analysis of thin sections of cells producing vesicles shows that a wide size range of vesicles are produced. The smallest vesicles observed measure approximately 0 ·1 μm in diameter. The great majority of vesicles, however, measure from 1 to 10 μm in diameter.

Fig. 3.

Transmission electron micrograph of SV3T3 cells showing the formation of a plasma membrane vesicles. This specimen was prefixed in calcium permanganate. × 10800.

Fig. 3.

Transmission electron micrograph of SV3T3 cells showing the formation of a plasma membrane vesicles. This specimen was prefixed in calcium permanganate. × 10800.

Morphology of isolated plasma membrane vesicles

By phase-contrast microscopy isolated plasma membrane vesicles have a spherical shape (Fig. 4A). By transmission electron microscopy plasma membrane vesicles also appear generally spherical or oval and are bordered by a unit membrane. The vesicles are free of contamination with cytoplasmic organelles (Fig. 4B) but do contain soluble cytosol components. When vesicles are examined by negative staining they appear to be quite polymorphous with numerous surface projections. Freeze-fracture analysis of isolated 3T3 and SV3T3 vesicles show that both contain intramembranous particles with a density similar to that present on intact cells (data not shown).

Fig. 4.

Phase-contrast micrograph (A) and a transmission electron micrograph (B) of isolated 3T3 plasma membrane vesicles, A, ×290;B, × 40000.

Fig. 4.

Phase-contrast micrograph (A) and a transmission electron micrograph (B) of isolated 3T3 plasma membrane vesicles, A, ×290;B, × 40000.

Enzyme characterization

Table 3 presents data which show that plasma membranes isolated from 3T3 and SV3T3 cells contain 5’nucleotidase activity which is enriched approximately 10-fold above that observed in whole cell homogenates. It also shows that plasma membrane preparations isolated from 3T3 or SV3T3 cells contain undetectable levels of NADH cytochrome C reductase and succinic dehydrogenase activity. The lower limits of detection in these assays were approximately 1 to 5 nmol cytochrome C reduced/min/ mg protein and 0·3 to 1 ·0 nmol indophenol reduced/min/mg protein, respectively.

Table 3.

Enzyme activities in isolated 3T3 and SV3T3 plasma membrane vesicles

Enzyme activities in isolated 3T3 and SV3T3 plasma membrane vesicles
Enzyme activities in isolated 3T3 and SV3T3 plasma membrane vesicles

Attempts to detect DNA in isolated plasma membrane vesicles were also made. DNA could not be detected. Initial studies employed the colorimetric diphenylamine procedure wherein the assay could detect 0·1 μg DNA. In subsequent studies vesicles were isolated from cells prelabelled with [3H] thymidine. The results of these assays showed that trichloroacetic acid-precipitated and alkahne-washed plasma membrane contained no detectable DNA.

These data estabfish that vesicles isolated by this procedure represent plasma membranes in that they show an ∼ 10-fold enrichment in 5’nucleotidase, a valid enzyme marker for fibroblast plasma membranes (Bingham & Burke, 1972; Perdue & Sneider, 1970) and no detectable contamination with marker enzymes for endoplasmic reticulum and mitochondria.

Plasma membrane vesicle protein characterization

When plasma membrane vesicles are lysed and washed, pure plasma membrane preparations can be prepared. Such specimens are free of soluble cytosol components. 3T3 and SV3T3 plasma membranes prepared in this manner contain approximately 20 polypeptides (Fig. 5). All stainable material entered 5 % gels. The results show that although minor and slightly variable differences in peak heights were observed between 3T3 and SV3T3 plasma membranes, no difference in the number of polypeptides nor in their molecular weight was detected by the procedure used in these studies.

Fig. 5.

Densitometric tracing of 3T3 (top) and SV3T3 (bottom) plasma membranes analysed by polyacrylamide gel (5 %) electrophoresis showing the presence of approximately 20 different size class polypeptides. Gels were stained with Coomassie blue.

Fig. 5.

Densitometric tracing of 3T3 (top) and SV3T3 (bottom) plasma membranes analysed by polyacrylamide gel (5 %) electrophoresis showing the presence of approximately 20 different size class polypeptides. Gels were stained with Coomassie blue.

Vesicle agglutination by plant lectins

Previous studies on intact 3T3 and SV3T3 cells showed that interphase 3T3 cells are less significantly agglutinated by Con A and WGA, than SV3T3 cells (Inbar & Sachs, 1969). Table 4 demonstrates somewhat similar characteristics of 3T3 and SV3T3 plasma membrane vesicles. Specimens not exposed to lectins showed no tendency to agglutinate; however, 3T3 vesicles showed slight agglutination with 10 μg/ml Con A and with 10-100 μg/ml WGA. SV3T3 vesicles showed marked agglutination following incubation in 10 μg/ml Con A and 100 μg/ml WGA. Exposure to 10μg/ml Con A or 100 μ g/ml WGA agglutinated essentially 100% of SV3T3 vesicles.

Table 4.

Agglutination of 3T3 and SV3T3 vesicles by plant lectins

Agglutination of 3T3 and SV3T3 vesicles by plant lectins
Agglutination of 3T3 and SV3T3 vesicles by plant lectins

Plasma membrane vesicle lipid characteristics

The results of studies to characterize the composition of 3T3 and SV3T3 plasma membrane lipids are given on Tables 5 and 6. In general, no major differences in lipid compositions were detected in these membrane preparations. The data, however, do show that plasma membrane preparations contain a much higher molar ratio of cholesterol: phospholipid than whole cell homogenates. Plasma membrane cholesterol/ phospholipid ratio was approximately 0·8 whereas that observed in 3T3 and SV3T3 whole cell homogenates was typically less than 0 · 3. Besides showing enrichment in cholesterol content, 3T3 and SV3T3 plasma membranes also showed an enrichment in sphingomyelin content. These findings are characteristic of plasma membrane preparations in general (Emmelot, Box, Van Hoeven & Van Blitterswijk, 1974).

Table 5.

Quantitation of vesicle lipid

Quantitation of vesicle lipid
Quantitation of vesicle lipid
Table 6.

Composition of 3T3 and SV3T3 vesicle phospholipid

Composition of 3T3 and SV3T3 vesicle phospholipid
Composition of 3T3 and SV3T3 vesicle phospholipid

In 1919 Hogue first reported that cell surface blebs were formed following exposure of cells to formaldehyde (Hogue, 1919). Landav & McAlene (1961) reported that high hydrostatic pressure could also produce blebs in some cells and in 1948 Zollinger described cell surface bleb formation and suggested that it resulted from submembrane swelling associated with the accumulation of water. Belkin & Hardy (1953) concurred and reported that sulphydryl blocking agents, including compounds containing mercury and arsenic, could induce the formation of blebs on cells.

More recent studies have described drug-induced membrane BLebbing (Godman, Meranda, Deitch & Tanenbaum, 1975; Nicolson, Smith & Poste, 1976), and the spontaneous formation of blebs in mitotic cell populations (Porter, Prescott & Frye, 1973). Trump’s studies suggested that most blebs form in response to cell injury (Trump, Croslev & Mergner, 1971). Although the formation of blebs resulting from cell injury may be associated with cell death, the formation of blebs did not in itself cause cell death (Trump et al. 1971).

Despite these numerous studies on cell surface BLebbing, previous reports have failed to show that there is significant shedding of blebs. Uhr, Vitetta & Melcher (1974) suggested that lymphocyte membrane antigens and antigen-antibody complexes might occur by the shedding of submicroscopic plasma membrane fragments but did not publish data to support this hypothesis. Studies on human red blood cells did demonstrate the shedding of small cell surface blebs in certain pathological conditions (Jacob, Overland, Ruby & Mazia, 1970; Reed & Swisher, 1966) and following exposure of red blood cells to the calcium ionophore A23187 (Allan, Billah, Finean & Michell, 1976).

In this paper we have presented evidence which shows that sulphydryl blocking agents can induce the shedding of cell surface vesicles from nucleated cells, such as fibroblasts. We have presented evidence that pure plasma membranes can be isolated using this technique. It has major advantages over other more traditional membrane isolation procedures. Our technique is simple, rapid, easily reproducible and produces a good yield of purified plasma membranes. Another advantage is that cells are not homogenized or disrupted by the process of vesiculation and, therefore, the chance of contamination with intracellular membranes is greatly reduced. Our data suggest that such plasma membrane preparations are representative of the cell surface, although we cannot exclude the possibility that minor differences might exist. The major disadvantage of the technique is that the vesiculants may inhibit some membrane enzymes.

Plasma membranes isolated by this technique can be used to study many important membrane characteristics. We have employed plasma membrane vesiculation techniques to isolate virus-free plasma membranes containing oncogenic Herpes virus specific membrane antigens (Pearson & Scott, 1977). We have also demonstrated that these plasma membrane preparations can be employed successfully as a virus-free immunogen to vaccinate susceptible hosts against a challenge with oncogenic Herpes virus.

In other studies these plasma membrane preparations have been employed to study the cyclic AMP-dependent phosphorylation of specific membrane polypeptides. The results demonstrated that cAMP-dependent protein kinase is present in plasma membranes isolated from L 6 myoblasts and from 3T3 and SV3T3 cells and that this cAMP-dependent protein kinase catalyses the phosphorylation of endogenous plasma membrane proteins (Scott & Dousa, 1978).

These and other data suggest that this new method for the isolation of plasma membranes should facilitate studies on the pathobiology of the plasma membrane in cultured mammalian cells.

This work was supported in part by the Mayo Foundation and by a Biomedical Research Grant no. 5 Sot RR 05530-14 and by a grant from the National Cancer Institute.

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