plasma membrane vesiculation in 3t3 and sv3t3 cells · the cell surface of normal and transformed...

J. Cell Sci. 35, 229-243 (1979) 229Printed in Great Britain © Company of Biologists Limited 1979

PLASMA MEMBRANE VESICULATION IN

3T3 AND SV3T3 CELLS

I. MORPHOLOGICAL AND BIOCHEMICALCHARACTERIZATION

ROBERT E. SCOTT, ROGER G. PERKINS,MICHAEL A. ZSCHUNKE, BRYAN J. HOERL ANDPETER B. MAERCKLEINLaboratory of Membrane Pathology, Department of Pathology and Anatomy,Mayo Clinic, Rochester, Minnesota 55901, U.S.A.

SUMMARY3T3 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 includingformaldehyde and AT-ethyl malemide. Morphological studies show that multiple vesicles areformed and released from individual cells and that the vesicle membrane is continuous with theplasma membrane of the cell. Vesicles measure from o-i to 15 /tm in diameter and are free ofdetectable contamination with cytoplasmic membranes and organdies. Vesicles also showa 1 o-fold enrichment in the plasma membrane marker enzyme 5'nucleotidase and are devoidof detectable NADH-cytochrome C reductase and succinic dehydrogenase activity which aremarker enzymes for endoplasmic reticulum and mitochondria, respectively. Vesicles havea high cholesterol: phospholipid ratio and show enrichment in sphingomyelin content. Theycontain receptors for Con A and WGA, approximately 20 size class polypeptides and intra-membranou8 particles. These results suggest that vesicles are derived from and have the generalcharacteristics of plasma membranes.

INTRODUCTION

The cell surface of normal and transformed cells has been studied in detail. Differ-ences 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 compositionof glycoproteins and glycolipids (Hynes, 1976) have been reported and it has beensuggested that there are differences in the interaction of the plasma membrane andthe microskeleton (Edelman, 1976). These data for the most part have been obtainedfrom studies using intact whole cells, whole cell homogenates or relatively crudemicrosomal-membrane fractions. Very few analytical studies have been performed onhighly purified plasma membranes of cells grown in tissue culture. An importantreason 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 ofplasma membrane vesicles from monolayer tissue culture cell lines, in particular 3T3and 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.

230 R. E. Scott and others

of sulphydryl blocking reagents induce the formation and release of vesicles from theplasma membrane (Scott, 1976). Vesicles released into the medium are free ofcontamination with cytoplasmic membranes but do contain soluble cytosol com-ponents which can be easily removed by lysis and washing of the vesicles to yieldpreparations of pure plasma membrane. Such specimens provide a new and valuablesource of material to study the compositional and structural characteristics of theplasma membrane.

MATERIALS AND METHODS

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 essentialmedium containing 10% calf serum (Grand Island Biological Co., Grand Island, N.Y.) at37 °C in a humidified atmosphere containing 10 % CO8 in air. Antibiotics were not addedto the medium and no evidence of fungal or bacteriological contamination was observed incells used in these studies. These cells were also tested and were found to be negative formycoplasma contamination by electron microscopy and cultural analysis. The saturationdensity for 3T3 cells used in these studies was approximately 4 x io* cells/cm1 and for SV3T3cells 2 x io6 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; L, myoblasts by Dr David Shubert;neuroblastoma cells (N1300) by Dr Elliot Richelson and owl monkey kidney cells by Dr GaryPearson. Human leukemic monocytes ( J -m) were purchased from the American Type TissueCulture Collection (ATCC). Guinea-pig peritoneal exudate macrophages were produced bysterile peritoneal irritation with Marcol 50 oil. Secondary cultures of low passage human skinfibroblasts were also used.

Plasma membrane vesicles

Plasma membrane vesiculation was tested by a standard method. Monolayer cell cultures weregrown to approximately 70% confluence in 60-mm Petri dishes (Falcon; Oxnard, CA) or onglass coverslips. Cells were washed 3 times in isotonic phosphate-buffered saline (pH 74)containing 0-75 min CaClj and 0-5 mM MgClj (CMPBS). Cells grown in suspension weresedimented by centrifugation at 100 g for 15 min and then washed 3 times in CMPBS. Cellswere then incubated in specific plasma membrane vesiculants. Vesiculation was scored ona scale of zero to four.

Bulk isolation of plasma membrane vesicles employed incubations of cells for 2 h at 37 CCin a vesiculant of 25 mM formaldehyde-2 mM dithiothreitol (DTT) prepared in phosphate-buffered saline containing 0-75 mM calcium and 05 mM magnesium (CMPBS). Vesicles weredecanted from the cells and passed through a glass wool column to remove detached cellaggregates. This solution was sedimented by centrifugation at 30000 g for 30 min at 4 °C. Theresulting translucent pellet was gently resuspended so as to leave any residual debris as a smalldense white pellet at the bottom of the tube. Vesicles were washed 3 times in 50 mM Tris-buffered saline, pH 7-4, and finally sedimented at 30000 g for 30 min at 4 °C. This plasmamembrane 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, RosebroughFarr & Randall (1951) and fluorescamine (Bohlen, Stein, Dairman & Udenfriend, 1973)procedures. Bovine serum albumin (BSA) was employed as the standard. Comparable resultswere obtained with both assays.

Plasma membrane vesiculation 231

Enzymatic characterization

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

The virtual absence of DNA in vesicles was demonstrated by the diphenylamine procedure(Burton, 1956) and by the demonstration that no detectable quantity of ['H]thymidine waspresent in TCA-precipitated membranes isolated from cells pulsed for 16 h in ['H]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 •$ % glutaralde-hyde - o-i M cacodylate buffer (pH 7-4) at room temperature for 1 h. They were then washed3 times in 01 M phosphate buffer (pH 7-4) and postfixed in 2 % osmium tetroxide for 30 minat room temperature. In some experiments specimens were prefixed in 2 % calcium per-manganate for 30 min at room temperature or fixed in glutaraldehyde containing 8 % tannicacid.

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

Specimens for scanning electron microscopy were routinely fixed in 2-5 % glutaraldehyde —o-i M phosphate buffer (pH 7-4) for 1 h at room temperature and then for 12 h at 4 °C. Somespecimens as described above were prefixed in 2 % calcium permanganate prior to glutaralde-hyde treatment. Fixed specimens were washed in o-i M phosphate (pH 7-4), dehydrated andcritical-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 examinedwith an ETEC stereoscan electron microscope operating at 20 kV.

Poly aery lamide gel electrophoresis

Purified plasma membranes were prepared from vesicle suspensions by addition of 50 mosmolbuffer followed by lysis by nitrogen cavitation (1-725 x ioa kN m"1 for 5 min). The membraneswere then collected by sedimentation at 250000 g for 1 h. Thereafter the membranes werewashed once and resedimented. This procedure releases soluble cytoplasmic proteins trappedwithin the vesicles. Purified plasma membrane pellets were solubilized in a solution containing1 % SDS-io mM DTT in o-oi M PO4 bufier at pH 7-4. This suspension was heated in a boilingwater bath for 5 min. Urea was added to a final concentration of 4 to 6 M and this solution wasthen sedimented at 20000 g for 15 min at room temperature to remove debris and any insolublematerial that was present. Known quantities of protein (25 to 75 fig) 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 Fair-banks, Steck & Wallach (1971). Coomassie blue-stained gels were scanned with a BeckmanActa II spectrophotometer equipped with a linear transport device. The molecular weights ofindividual polypeptides were calibrated using f3 galactosidase, bovine serum albumin andcytochrome C as standards.

Lectin-induced vesicle agglutination

The agglutinability of plasma membrane vesicles derived from 3T3 and SV3T3 cells withConcanavalin A (Con A) and wheat germ agglutinin (WGA) was tested. Con A was purchasedfrom 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. Vesicleswere then sedimented by centrifugation at 30000 g for 30 min at room temperature and gentlyresuspended. The degree of vesicle agglutination for control and lectin-exposed samples wasdetermined by phase microscopy. Vesicle agglutination was scored from o to + + + + .Agglutination of + + + + was scored when > 75 % of the vesicles agglutinated; + + +,> 5O%; + +, > 25 %, and +, < 25 %.

Phospholipid and cholesterol analysis

The lipid composition of 3T3 and SV3T3 whole cell homogenates and 3T3 and SV3T3plasma membrane vesicles was determined by extraction of aqueous membrane pellets withcold chloroform: methanol (2:1) according to the procedure described by Perkins & Kummerow(1976). Free cholesterol and total phospholipid were determined using modified versions ofthe procedures reported by Searcy & Berquist (i960) and Eng & Noble (1968), respectively.

Reagents

All reagents were purchased from commercial sources in the highest grade available. Formal-dehyde was prepared from paraformaldehyde (Aldrich Chemical Co., Milwaukee, WI) bydissolving 50 g in 500 ml HjO. This solution was heated at 60 °C for 1 h then NaOH (1 M) wasadded dropwise until the turbid solution cleared. This 10 % formaldehyde solution was pre-pared at weekly intervals and was stored at 4 °C under nitrogen after filtration through What-man no. 1 filter paper.

RESULTS

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, 1966a, b) induce plasma membrane vesiculation in

Table 1. Effective plasma membrane vesiculants in 3 73 cells

Vesiculants

FormaldehydePyruvic aldehydeAcetaldehydeGlyoxalGlutaraldehydeAcroleinMethacroleinPyridoxalN-ethyl malemideMalemide/>-ChloromercuribenzoateIodoacetate

Minimumeffective

concentration

250 min01 %

1 %

1 %001 %

o - i %o - i %

10 rriM10 mM

1 mM1 mM

Maximumvesiculation*

+ + + ++ ++ ++ ++

+ /+ ++ /+ +

+ ++ + + +

+ ++ /+ +

+ +• Results obtained on 3T3 cells. All reagents were prepared in CMPBS, and incubations were

at 37 °C. Vesiculation is scored on a scale of—to + + + + by phase microscopy. Quantitationof this scoring procedure on the basis of vesicle protein released is reported in the text. Theminimum effective vesiculant concentration could be reduced for some compounds whendithiothreitol was added.


cultured mammalian cells (Table 1). Formaldehyde, and iV-ethyl malemide were ingeneral the most effective vesiculants. No observable vesiculation could be detectedwith alcohols, acids, ketones, or with succinimide, a maleimide derivative which doesnot bind to free sulphydryl groups (Webb, 1966 a, b). Reagents which only reducedisulphide bonds, such as dithiothreitol, 2-mercaptoethanol, and L-cysteine, also didnot induce vesiculation. Table 2 shows that the phenomenon of plasma membranevesiculation was observed in a wide variety of monolayer cell cultures. These includefibroblasts, myoblasts, macrophages and cells of neural origin. Aldehyde-inducedvesiculation was observed in cell lines of mouse, rat, guinea pig, monkey and humanorigin; in primary and secondary cultures and in established cell lines. These variouscells all grew as monolayer cultures. The plasma membrane vesiculation techniqueis not effective for suspension cultures, at least under the conditions used in theseexperiments.

Table 2. Effect of a plasma membrane vesiculant on different cell populations

Maximum plasmamembrane

Monolayer cell cultures Species vesiculation*

Balb/c 3T3Swiss 3T3AKR embryo cellsSV3T3MCA 3T3AKR-MCAXC CellsLeukemic monocytes ( J -m)Peritoneal macrophagesL, myoblastsKidney (OMK)Herpes virus saimiri-infected OMKNeuroblastoma cells (N1300)Secondary cultures of skin fibroblasts

MouseMouseMouseMouseMouseMouseRatHumanGuinea pigRatOwl monkeyOwl monkeyMouseHuman

• Vesiculation induced with 25 mM formaldehyde-2 mM dithiothreitol in CMPBS byincubation at 37 °C. Vesiculation was scored at approximately 2 h.

Morphological characteristics of vesiculation in 3 T3 and SVy T3 cells

Fig. 1 illustrates plasma membrane vesiculation in 3T3 and SV3T3 cells. Un-treated 3T3 cells show a polygonal cell shape with little cell overlapping and no evi-dence of vesiculation (Fig. IA). Following exposure to formaldehyde for 15 min,minute optically dense spherical protrusions appear on the cell surface. These smallblebs enlarge and after incubation for 30 min numerous 0 1 - to io-/tm projections canbe seen. The formation and release of vesicles progresses rapidly at 37 °C and after 60to 90 min large quantities of cell surface vesicles can be seen in the medium (Fig. 1 B).The process of vesiculation continues for approximately 2 h. Fig. ic and D comparethe appearance of native SV3T3 cells and those shedding vesicles. The morphological •appearance of cells incubated in other vesiculants was comparable.

Fig. 2 illustrates plasma membrane vesicle formation in 3T3 and SV3T3 cells by

R. E. Scott and others

>a • B .

Mm• - • < '

Fig. i. Phase-contrast micrographs of 3T3 (A, B) and SV3T3 (c, D) mouse embryocells: before (A, C) and after (B, D) the formation and shedding of plasma membranevesicles, A, X 195; B, X205; c, X210; D, X 195.


Fig. 2. Scanning electron micrographs of 3T3 (A) and SV3T3 cells (B, C) formingplasma membrane vesicles. A, x 1760; B, x 2400; c, x 1560.Fig. 3. Transmission electron micrograph of SV3T3 cells showing the formation ofa plasma membrane vesicles. This specimen was prefixed in calcium permanganate,x 10800.


«•'••• • . .

f

V.V

Fig. 4. Phase-contrast micrograph (A) and a transmission electron micrograph (B) ofisolated 3T3 plasma membrane vesicles, A, X29O;B, x 40000.


scanning electron microscopy. Vesicles initially develop at the cell margin and appearas small focal swellings (Fig. 2 A, B). Subsequently, vesicles are formed and releasedfrom different regions of the cell surface. Up to ten plasma membrane vesicles havebeen observed to arise from an individual cell. In many cases vesicles of different sizesand of different sites of origin can be observed on an individual cell (Fig. 2 B) and insome cases small projections are observed on the vesicle itself (Fig. 2 c). These smallprojections are also observed in negatively stained preparations of isolated vesicles.

By transmission electron microscopy it is apparent that vesicles develop as saccularoutpouching of the plasma membrane (Fig. 3). The vesicles show a unit membranewhich is continuous with the plasma membrane of the cell. The cytoplasmic surfaceof the plasma membrane vesicle contains adherent fibrous material and cytosol butno organelles and very few polysomes. Ultrastructural analysis of thin sections of cellsproducing vesicles shows that a wide size range of vesicles are produced. The smallestvesicles observed measure approximately o-i /tm in diameter. The great majority ofvesicles, however, measure from 1 to 10 fim in diameter.

Morphology of isolated plasma membrane vesicles

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

Enzyme characterization

Table 3 presents data which show that plasma membranes isolated from 3T3 andSV3T3 cells contain 5'nucleotidase activity which is enriched approximately 10-foldabove that observed in whole cell homogenates. It also shows that plasma membranepreparations isolated from 3T3 or SV3T3 cells contain undetectable levels of NADHcytochrome C reductase and succinic dehydrogenase activity. The lower limits ofdetection in these assays were approximately 1 to 5 nmol cytochrome C reduced/min/mg protein and 0-5 to i-o nmol indophenol reduced/min/mg protein, respectively.

Attempts to detect DNA in isolated plasma membrane vesicles were also made.DNA could not be detected. Initial studies employed the colorimetric diphenylamineprocedure wherein the assay could detect o-1 fig DNA. In subsequent studies vesicleswere isolated from cells prelabelled with [3H]thymidine. The results of these assaysshowed that trichloroacetic acid-precipitated and alkaline-washed plasma membranecontained no detectable DNA.

These data establish that vesicles isolated by this procedure represent plasmamembranes in that they show an ~ 10-fold enrichment in 5'nucleotidase, a validenzyme marker for fibroblast plasma membranes (Bingham & Burke, 1972; Perdue &

16 CHL 35


Table 3. Enzyme activities in isolated ^TT, and SV^Ti, plasma membrane vesicles

3T3Whole cellsPF/DTT-treated whole cellsPlasma membrane vesiclesEnrichment

SV3T3Whole cellsPF/DTT-treated whole cellsPlasma membrane vesiclesEnrichment

Enzyme activities are reported as

sTSIucleo-tidase,nmol

Pi released/min/mgprotein

2-4±o-63-3 ±1-0

23-5 ±i -69-8X

6-i ±0-96-6±o-6

55-6U-89-iX

NADHcyto-

chrome Creductase,

nmolcyto-

chrome Creduced/min/mgprotein

4i-S±430-0 ±3

< i-o

59-0 ±223-0 ± 1

< i-o•

Succinicdehydro-genase,nmol

indophenolreduced/min/

mg protein

4-0 ±0-54-0 ±0-4

< o-s

6-4±o-i3'5 ±°-3

< o-s

DNA,

DNA/mgprotein

88-o

ND

995

ND

mean values ± standard error of mean. ND, not detectable.

Sneider, 1970) and no detectable contamination with marker enzymes for endoplasmicreticulum and mitochondria.

Plasma membrane vesicle protein characterization

When plasma membrane vesicles are lysed and washed, pure plasma membranepreparations can be prepared. Such specimens are free of soluble cytosol components.3T3 and SV3T3 plasma membranes prepared in this manner contain approximately20 polypeptides (Fig. 5). All stainable material entered 5 % gels. The results show thatalthough minor and slightly variable differences in peak heights were observed between3T3 and SV3T3 plasma membranes, no difference in the number of polypeptides norin their molecular weight was detected by the procedure used in these studies.

Vesicle agglutination by plant lectins

Previous studies on intact 3T3 and SV3T3 cells showed that interphase 3T3 cellsare less significantly agglutinated by Con A and WGA, than SV3T3 cells (Inbar &Sachs, 1969). Table 4 demonstrates somewhat similar characteristics of 3T3 andSV3T3 plasma membrane vesicles. Specimens not exposed to lectins showed notendency to agglutinate; however, 3T3 vesicles showed slight agglutination withio/tg/ml Con A and with 10-100/ig/ml WGA. SV3T3 vesicles showed markedagglutination following incubation in 10 //.g/ml Con A and 100 /tg/ml WGA. Exposureto 10/ig/ml Con A or 100/ig/ml WGA agglutinated essentially 100% of SV3T3vesicles.


1 i

134000 68000 24000

Mol. wt., Daltons

12000

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

Table 4. Agglutination of 3 ^ and SV^TT, vesicles by plant lectins

3T3 PMV SV3T3 PMV

ControlCon A, /Jg/ml

WGA, jig/ml

i10

ICO

I

10IOO

16-2


Plasma membrane vesicle lipid characteristics

The results of studies to characterize the composition of 3T3 and SV3T3 plasmamembrane lipids are given on Tables 5 and 6. In general, no major differences inlipid compositions were detected in these membrane preparations. The data, however,do show that plasma membrane preparations contain a much higher molar ratio ofcholesterol: phospholipid than whole cell homogenates. Plasma membrane cholesterol/phospholipid ratio was approximately o-8 whereas that observed in 3T3 and SV3T3

Table 5. Quantitation of vesicle lipid

3T3 PMV SV PMV

Phospholipid, /ttnol/mg proteinCholesterol, /wnol/mg proteinCholesterol/phospholipid, molar ratioPhospholipid, fig/fig proteinCholesterol, fig/fig protein

Table 6. Composition 0/3T3 and SF3T3 vesicle phospholipid

Phospholipid composition, %

0229O-I7S07640177

0-067

0243O-2OS

084601880-078

Phosphatidyl cholinePhosphatidyl ethanolaminePhosphatidyl serinePhosphatidyl inositolSphingomyelinOthers

Whole

3T3

5429

7334

cells

SV3T3

5822

235

10

Plasma membranevesicles

3T3

541776

16

SV3T3

59146

1011

whole cell homogenates was typically less than 0-3. Besides showing enrichment incholesterol content, 3T3 and SV3T3 plasma membranes also showed an enrichmentin sphingomyelin content. These findings are characteristic of plasma membranepreparations in general (Emmelot, Box, Van Hoeven & Van Blitterswijk, 1974).

DISCUSSION

In 1919 Hogue first reported that cell surface blebs were formed following exposureof cells to formaldehyde (Hogue, 1919). Landav & McAlene (1961) reported that highhydrostatic pressure could also produce blebs in some cells and in 1948 Zollingerdescribed cell surface bleb formation and suggested that it resulted from submembraneswelling associated with the accumulation of water. Belkin & Hardy (1953) concurredand reported that sulphydryl blocking agents, including compounds containingmercury 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 thespontaneous 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 fromcell injury may be associated with cell death, the formation of blebs did not in itselfcause cell death (Trump tt al. 1971).

Despite these numerous studies on cell surface blebbing, previous reports havefailed to show that there is significant shedding of blebs. Uhr, Vitetta & Melcher(1974) suggested that lymphocyte membrane antigens and antigen-antibody com-plexes might occur by the shedding of submicroscopic plasma membrane fragmentsbut did not publish data to support this hypothesis. Studies on human red blood cellsdid demonstrate the shedding of small cell surface blebs in certain pathologicalconditions (Jacob, Overland, Ruby & Mazia, 1970; Reed & Swisher, 1966) andfollowing 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 blockingagents can induce the shedding of cell surface vesicles from nucleated cells, such asfibroblasts. We have presented evidence that pure pla&ma membranes can be isolatedusing this technique. It has major advantages over other more traditional membraneisolation procedures. Our technique is simple, rapid, easily reproducible and producesa good yield of purified plasma membranes. Another advantage is that cells are nothomogenized or disrupted by the process of vesiculation and, therefore, the chance ofcontamination with intracellular membranes is greatly reduced. Our data suggestthat such plasma membrane preparations are representative of the cell surface,although we cannot exclude the possibility that minor differences might exist. Themajor disadvantage of the technique is that the vesiculants may inhibit somemembrane enzymes.

Plasma membranes isolated by this technique can be used to study many import-ant membrane characteristics. We have employed plasma membrane vesiculationtechniques to isolate virus-free plasma membranes containing oncogenic Herpes virusspecific membrane antigens (Pearson & Scott, 1977). We have also demonstrated thatthese plasma membrane preparations can be employed successfully as a virus-freeimmunogen to vaccinate susceptible hosts against a challenge with oncogenicHerpes virus.

In other studies these plasma membrane preparations have been employed to studythe cyclic AMP-dependent phosphorylation of specific membrane polypeptides. Theresults demonstrated that cAMP-dependent protein kinase is present in plasmamembranes isolated from L6 myoblasts and from 3T3 and SV3T3 cells and that thiscAMP-dependent protein kinase catalyses the phosphorylation of endogenousplasma membrane proteins (Scott & Dousa, 1978).

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

242 R- E. Scott and others

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

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{Received 3 August 1977 - Revised 7 June 1978)

plasma membrane vesiculation in 3t3 and sv3t3 cells · the cell surface of normal and transformed...

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