J. CellSci. 36,61-72 (1979) 61Printed in Great Britain (c) Company of Biologist! Limited 1 oyg

STUDIES OF MEMBRANE FUSION.

III. FUSION OF ERYTHROCYTES WITHPOLYETHYLENE GLYCOL

S. KNUTTONDepartment of Biochemistry, St George's Hospital Medical School,Cranmer Terrace, London SWi"j oKE, England

SUMMARY

Freeze-fracture electron microscopy has been used to investigate the mechanism of poly-ethylene glycol-induced cell fusion. Interaction of cells with the high concentrations of poly-ethylene glycol required for cell fusion results in cell agglutination with large planar areas ofvery close contact between adjacent cell membranes. An aggregation of intramembraneparticles into large patches at the sites of cell-cell contact accompanies cell agglutination.Fusion occurs following the removal of most of the PEG when cells only remain in closecontact at small (~ 01 fim diameter) plaques of smooth particle-denuded membrane. Mem-brane fusion occurs at these plaques of smooth membrane resulting in cells connected by one(or more) small cytoplasmic connexions. Expansion to form spherical fused cells occurs by aprocess of cell swelling.

INTRODUCTION

High-molecular-weight polymers of polyethylene glycol (PEG) were first shown tobe potent fusogens for plant protoplasts (Kao & Kichayluk, 1974; Wallin, Glimelius &Erikkson, 1974) but more recently have been used to fuse a variety of cell types(Ahkong et al. 19756; Pontecorvo, Riddle & Hales, 1977). Because PEG is relativelynon-toxic, yields a high incidence of cell fusion and can be used to fuse a wide varietyof different cells including interspecific and interkingdom cell types (Ahkong et al.19756; Jones et al. 1976), it is rapidly becoming a very widely used cell fusogen. Mostof the studies, however, have emphasized techniques for optimizing the production ofcell hybrids; few studies have been concerned with the actual mechanism of PEG-induced cell fusion (Maggio, Ahkong & Lucy, 1976; Maul, Steplewski, Weibel &Koprowski, 1976). Thin-section observations (Maul et al. 1976) have shown thatcell-cell fusion initially occurs at small localized areas of the cell surface but, becauseof the limited information present in images of cross-sectioned membranes, thesestudies have provided little insight into the actual membrane modifications takingplace which allow membranes to fuse. The freeze-fracture technique, on the otherhand, allows one to examine extensive en face views of membranes and also has theproperty of allowing integral membrane proteins and phospholipids to be distin-guished morphologically and, therefore, offers the opportunity of defining how thesedifferent membrane components are involved in membrane fusion. In this paper Ipresent freeze-fracture observations on the fusion of human erythrocytes and describe

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62 S. Knutton

PEG-induced changes in membrane structure which show that membrane fusionoccurs between small localized areas of naked lipid bilayer. Subsequent cell swellingresults in the production of spherical fused cells.

METHODS

Cell fusion

PEG-induced fusion of erythrocytes was carried out essentially according to the procedureof Davidson & Gerald (1977). Human or chick erythrocytes were washed 3 times in Hanks'balanced salt solution (HBSS) (Hanks & Wallace, 1949) and a 2% suspension (~i '5Xio8

cells/ml) used for fusion experiments. 1 ml of an erythrocyte suspension was centrifuged(1000 g, S min) and the cell pellet resuspended in 1 ml of a 50 % solution of PEG (6000 mole-cular weight) in HBSS at 4 °C. After 1 min the aggregated cells were diluted with 9 ml ofHBSS and the cells centrifuged (1000 g, 5 min). The cell pellet was resuspended in 5 ml offresh HBSS and fusion initiated by transferring the cell suspension to a waterbath at 37 °C.Samples of agglutinated cells (cells in 50 % PEG), following removal of most of the PEG at4 CC and cells incubated for up to 60 min at 37 °C, were processed for thin section, scanning andfreeze-fracture electron microscopy. Cell fusion was assessed by light microscopy.

Electron microscopy

For thin sections cells were fixed with 3 % glutaraldehyde in o-i M sodium cacodylate pH 74for 1 h, postfixed with 1 % buffered osmium tetroxide for 2 h, blockstained with 2 % aqueousuranyl acetate, dehydrated through a graded series of ethanol and propylene oxide solutionsand embedded in Epon. Sections were cut with glass knives on a Tesla ultramicrotome, stainedwith uranyl acetate and lead citrate and examined in a Siemens 101 electron microscope.

For scanning electron microscopy cells were allowed to settle on to gelatin-coated glasscoverslips (Vial & Porter, 1975) and immediately fixed with 3 % glutaraldehyde in HBSS for30 min. In some cases cells were postfixed for 1 h with 1 % buffered osmium tetroxide prior tobeing dehydrated through a graded series of acetone solutions. The coverslips were thentransferred to liquid carbon dioxide and critical-point dried. Finally the coverslips weremounted on aluminium stubs, coated with a thin layer of gold, and the specimens examined ina Cambridge Type II stereoscan.

For freeze-fracture electron microscopy cells were fixed with 3 % glutaraldehyde in HBSSfor 30 min, washed, and infiltrated with 25 % glycerol also in HBSS. Specimens were rapidlyfrozen in freshly melted Freon 22 and platinum carbon replicas made in a Denton freeze-fracture machine. Replicas were cleaned with bleach and washed in distilled water before beingpicked up on uncoated grids and examined. In freeze-fracture micrographs the encircledarrows indicate the direction of platinum shadowing.

OBSERVATIONS

Suspending cells in a 50% solution of PEG results in instantaneous cell aggluti-nation and the formation of large non-specific cell aggregates (Fig. IA). Cells shrinkand become highly distorted as a result of the formation of large planar areas of closecontact between cells (Figs. 1-3). At regions of close apposition adjacent cell mem-branes are separated by less than 5 nm and in many cases they come into sufficientlyclose contact that one sees an apparent fusion of the outer dense leaflets of the adjacent'unit membrane' structures (Fig. 2). However, at this stage, the 2 membranes are notfused in any strict sense of the term and removal of the PEG by washing results inseparation of the agglutinated cells. By freeze-fracture electron microscopy it can beseen that PEG treatment induces dramatic structural changes in the plane of the

Erythrocyte fusion with ethylene glycol

Fig. 1. Scanning (A) and freeze-fracture electron micrographs (B) showing humanerythrocytes agglutinated with 50 % PEG. Cells form large non-specific aggregateswith planar areas of close contact between adjacent cells. A, x 4800; B, X 1800.

Fig. 2. Cross-sections through ragions of contact between agglutinated erythrocytes.Adjacent membranes are separated by less than ~ 5 nm and in many areas there isfusion of the outer dense leaflets of adjacent 'unit membrane' structures, A, X 170000;B, x 136000.

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Fig. 3. Freeze-fracture replicas showing erythrocytes agglutinated with PEG. Planarregions of contact reveal a clustering of P-face intramembrane particles into smallpatches (A) or large aggregates (B). A complementarity exists between particulate andsmooth regions of adjacent membranes (B). P and E fracture faces not in contact withan adjacent cell still retain a random distribution of intramembrane particles (A,asterisks), A, x 22000; B, x 74000,

Erythrocyte fusion with ethylene glycol 65

membrane (Fig. 3). Fractures through agglutinated cell aggregates reveal the largeplanar areas of close cell contact (Fig. 3 A). PEG induces an aggregation of the Pfracture face intramembrane particles into small patches (Fig. 3 A) or large aggregates(Fig. 3 B) separated by islands of smooth membrane. Such particle aggregates,however, only occur at sites of cell-cell contact. Regions of cell membrane not incontact with an adjacent cell retain an essentially random distribution of membraneparticles (Figs. 1 B, 3 A, asterisks). The tight interaction between adjacent cell mem-branes at sites of contact is such that the fracture plane frequently jumps from onemembrane to the other (Fig. 3 A). At higher magnifications (Fig. 3B) it can be seen thatthere exists a complementarity between smooth and particulate regions of adjacent cellmembranes. This is readily apparent because an impression of the aggregated P-faceintramembrane particles can be seen on the E fracture face of the adjacent membrane.The aggregates of P-face particles can be seen to correspond to the aggregates of E-facepits; similarly there is a complementarity between smooth regions of fracture face. TheE-face intramembrane particles although unaggregated are also localized in regions offracture face associated with the aggregates of E face pits (Fig. 3B).

Following the removal of most of the PEG, cells remain agglutinated but are lesstightly bound. Rather than being in close contact over large areas of cell surface,oblique fractures through agglutinated cells show that cells only remain in closecontact at small (~ o-i /im diameter) plaques of smooth particle-denuded membrane(Fig. 4c, D, arrows). Intercellular space is clearly visible in between the plaques ofsmooth membrane. That adjacent cells remain tightly bound, however, is indicatedby the fact that the membrane becomes significantly distorted at these focal points ofcontact (Fig. 4D, arrows). In regions of fracture face no longer in close contact withan adjacent cell the intramembrane particles appear to have returned to an essentiallyrandom distribution (Fig. 4A, B).

Incubation of an agglutinated cell suspension from which most of the PEG hasbeen removed for 30-60 min at 37 °C results in extensive cell-cell fusion and theformation of large spherical polyerythrocytes (Fig. 7). Other unfused erythrocytes alsoswell (Fig. 7) and haemolysis of most cells eventually takes place. Intermediate stagesduring the formation of spherical fused cells are revealed by taking samples after brief(0-10 min) periods of incubation. The earliest fusion event appears to involve fusionbetween small areas of adjacent cell membrane resulting in cells connected by small(~ 0-1-0-2 /tm diameter) cytoplasmic connexions (Fig. 5 A, B). The appearance of theintracellular matrix at this stage (Fig. 5 A) indicates that the cells are still unswollenand are not haemolysed.

Light microscopy indicates that spherical fused cells arise from cells connected bysmall cytoplasmic bridges by a process of cell swelling during which the cytoplasmicbridges become expanded. Expansion of the initial cytoplasmic connexions duringswelling results in fusing cells having a dumbbell shape and many such cells are seenduring early stages of fusion. Fig. 6 shows one polyerythrocyte resulting from fusionof at least four individual erythrocytes which has not yet expanded to a spherical shape.The dumbbell shape of the fusing cells is readily apparent. The freeze-fractureappearances of fused cells are similar to controls; there is an essentially random

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Fig. 4. Freeze-fracture replicas showing erythrocytes following the removal of mostPEG. Cells only remain agglutinated at small (~o-i /tm diameter) plaques of smoothparticle-denuded membrane (arrows). The interaction of adjacent membranes at thefocal points of contact often distorts the membranes (c, D, arrows). Other regions offracture face display an essentially random distribution of intramembrane particles(A, B). A, X 45000; B, X 45000; C, X 39000; D, x 45000.

Erythrocyte fusion with ethylene glycol

Fig. 5. Freeze-fracture replicas showing erythrocytes fused at 37 CC for 5 min.Initially one sees cells (/, 2) connected by small (~ 0-1-0-2 fim diameter) cytoplasmiccontinuities (arrows). Other regions of close cell contact remain unfused (arrowheads).A, x 11 500; inset, x 80000; B, x 58000.

distribution of intramembrane particles (Fig. 6), although in many instances, PEGtreatment and the subsequent incubation at 37 °C has the effect of inducing segre-gation of smooth membrane (Fig. 6, asterisk) and the formation of droplets identifiedas lipid by their morphology. Such lipid droplets frequently bleb off from either theinterior or exterior cell surface (Fig. 6).

68 S. Knutton

Erythrocyte fusion with ethylene glycol 69

DISCUSSION

It has been proposed that cell-cell fusion occurs by a mechanism which involvesthree distinct stages (Knutton, 1978). Cell fusogens induce (1) close contact betweenadjacent cell membranes; (2) membrane fusion at small localized sites of cell contact;and (3) expansion of sites of membrane fusion to form spherical fused cells by a processof cell swelling. Although the molecular mechanisms whereby different fusogens elicitthis sequence of events clearly differ, the morphological observations presented hereand illustrated in diagrammatic form (Fig. 8) are consistent with such a mechanism.

PEG does induce cell agglutination and the formation of very close contactsbetween adjacent cell membranes. Normally, the plasma membranes of adjacent cellsvisualized by transmission electron microscopy do not approach each other closerthan ~ 20 nm. This is both because of mutual electrostatic repulsion between 2closely apposed membranes and because of the exclusion volume of the plasmamembrane glycoproteins and glycocalyx macromolecules (Maroudas, 1975). PEG mayfacilitate close cell contact (< 5 nm) by reducing the exclusion volume of membraneglycoproteins. In addition to facilitating close membrane contact PEG induces anaggregation of intramembrane particles. Once close apposition (i.e. < 5 nm) betweencharged membranes is achieved, an aggregation of intramembrane particles couldoccur by direct electrostatic displacement (Gingell & Ginsberg, 1978). In the case oferythrocytes it is known that the intramembrane particles are associated with the majorcharge-bearing groups of the cell surface (Pinto da Silva & Nicolson, 1974). Althoughother mechanisms of intramembrane particle aggregation are possible (Gingell &Ginsberg, 1978), e.g. spectrin aggregation (Elgsaeter, Shotton & Branton, 1976), thatcontact-mediated electrostatic displacement is the mechanism in this case is supportedby the observation that intramembrane particle aggregation occurs only at sites ofclose cell-cell contact and not over the remainder of the cell surface. Furthermore,removal of PEG reverses the process of particle aggregation. An aggregation ofintramembrane particles was suggested in the study of PEG-induced fusion of mouseL cells (Maul et al. 1976) but these authors failed to examine cells in the presence ofhigh concentrations of PEG. Although a direct correlation between thin-section andfreeze-fracture images cannot be made, it seems likely that the regions of membraneshowing very close contact (i.e. where there is an apparent fusion of the outer denseleaflets of adjacent 'unit membrane' structures) represent complementary regions ofintramembrane particle-denuded lipid bilayer since, on removal of most of the PEG,

Fig. 6. Freeze-fracture replicas showing the P-fracture face of a polyerythrocyte formedas the result of fusion of at least four individual erythrocytes (A) and part of the P faceof another fused cell (B). Segregation of smooth membrane is apparent (asterisks)and droplets (/) can be seen blebbing or to have blebbed off from the membrane.Elsewhere there is a normal distribution of intramembrane particles. A, x 13500;B, x 40000.

Fig. 7. Phase-contrast micrograph showing chick erythrocytes fused for 30 min at37 °C. Both fused and unfused cells are swollen, spherical, and at least partiallyhaemolysed. x 340.

70 S. Knutton

cells only remain in close contact at small plaques of smooth membrane and thin-section images show that fusion of the 'unit membrane' structures also occurs atthese plaques.

Fig. 8. Diagrammatic representation of stages during PEG-induced fusion oferythrocytes. Addition of 50 % PEG to cells (A) results in agglutination. Cells aredistorted to form large areas of close cell contact (B). Removal of most PEG leavescells agglutinated only at small plaques of smooth membrane (c). Warming to 37 CCinitiates membrane fusion at one (or more) of these plaques resulting in the formationof one (or more) cytoplasmic connexions (D). Expansion of small cytoplasmic bridgesduring cell swelling (E) produces a spherical fused cell (F).

In agreement with previous studies with mouse L cells (Maul et al. 1976), PEG-induced fusion of erythrocytes is also seen to occur initially at small localized areas.Since membranes remain in close contact only at small plaques of smooth intra-membrane particle-denuded membrane following removal of PEG and since thecytoplasmic continuities initially formed are the same order of magnitude it seemsreasonable to conclude that membrane fusion has occurred at the plaques of smoothmembrane. There is now a considerable body of evidence to suggest that membranefusion occurs via lipid-lipid interactions between protein-depleted regions of adjacentapposed membranes (Satir, Schooley & Satir, 1973; Papahadjopoulos, Poste &Shaeffer, 1973; Orci, Perrelet & Friend, 1977; Lawson et al. 1977) and a mechanismof fusion based on the interaction and fusion of naked lipid bilayers has been pro-posed (Ahkong, Fisher, Tampion & Lucy, 1975 a). The observations presented heresuggest that PEG-induced membrane fusion also occurs by such a mechanism.

The formation of a spherical fused cell, following membrane fusion at one (ormore) small localized areas of cell contact, must involve membrane redistribution and

Erythrocyte fusion toith ethylene glycol 71

expansion of the cytoplasmic continuities. Both light and electron microscopy suggestthat this occurs by a process of cell swelling. It is clear that erythrocytes fused at 37 °Cfor 30 min are all swollen and, in most cases, haemolysed; at earlier stages, fusing cellswith partially expanded cytoplasmic continuities are common. Biochemical studieshave shown that during PEG-induced cell fusion there is a change in membranepermeability (Knutton, Micklem & Pasternak, unpublished observations). Loss ofcation asymmetry, as is known to take place during Sendai virus-induced cell fusion(Poste & Pasternak, 1978), would then result in entry of water and cell swelling. Theorigin of the membrane permeability change is still unknown in the case of PEGtreatment but the freeze-fracture observations show that PEG does perturb membranestructure quite drastically, sometimes in an irreversible manner, and frequentlyresults in the segregation of lipids and the blebbing off of lipid droplets from the cells.Such membrane perturbations could lead to changes in membrane permeability. Onthe other hand, lipid segregation may be the consequence of changes in membranepermeability since it has been shown that conditions which cause a contraction of theactin-spectrin meshwork compress the lipid bilayer of the membrane causing it tobleb off particle-free vesicles (Elgsaeter et al. 1976). Entry of external Ca2+ forexample, could be one possible cause of lipid blebbing. Nevertheless, membranepermeability changes, whatever their origin, which result in cell swelling do appear toprovide the driving force which expands cells connected by small cytoplasmicconnexions to form spherical fused cells.

I am grateful to Mrs Diane Jackson for excellent technical assistance and the Cancer ResearchCampaign for financial assistance.

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{Received 8 August 1978)