,jf. Celt Sci. 7 , 3 i 9 - 3 3 S ( i 970) 319
Printed in Great Britain
THE RAPID INTERMIXING OF CELL SURFACE
ANTIGENS AFTER FORMATION OF MOUSE-
HUMAN HETEROKARYONS
L. D. FRYE* AND M. EDIDINf
Department of Biology, The Johns Hopkins University,
Baltimore, Maryland 21218, U.S.A.
SUMMARY
Cells from established tissue culture lines of mouse {CIID) and human (VA-2) origin were
fused together with Sendai virus, producing heterokaryons bearing both mouse and human
surface antigens which were then followed by the indirect fluorescent antibody method. Within
40 min following fusion, total mixing of both parental antigens occurred in over 90 % of the
heterokaryons.
Mouse H-2 (histocompatibility) and human surface antigens were visualized by successive
treatment of the heterokaryons with a mixture of mouse alloantiserum and rabbit anti-VA-2
antiserum, followed by a mixture of fluorescein-labelled goat anti-mouse IgG and tetramethyl-
rhodamine-labelled goat anti-rabbit IgG(Fc).
The CIID x VA-2 fusions were carried out in suspension and maintained at 37 CC in a shaking
water bath; aliquots were removed at various intervals and stained with the above reagents. The
heterokaryon population was observed to change from an initial one (5-min post-fusion) of
non-mosaics (unmixed cell surfaces of red and green fluorescence) to one of over 90 % mosaics
(total intermixing of the 2fluorochromes)by 40 min after fusion. Mouse-human hybrid lines,
derived from similar fusions, gave fluorescence patterns identical to those of the mosaic
heterokaryons.
Four possible mechanisms would yield such results: (i) a very rapid metabolic turnover of
the antigens; (ii) integration of units into the membrane from a cytoplasmic precursor pool;
(iii) movement, or ' diffusion 'of antigen in the plane of the membrane; or (iv) movement of exist-
ing antigen from one membrane site into the cytoplasm and its emergence at a new position on
the membrane.
In an effort to distinguish among these possibilities, the following inhibitor treatments were
carried out: (1) both short- and long-term (6-h pre-treatment) inhibition of protein synthesis
by puromycin, cycloheximide, and chloramphenicol; (2) short-term inhibition of ATP forma-
tion by dinitrophenol (DNP) and NaF; (3) short- and long-term inhibition of glutamine-
dependent pathways with the glutamine analogue 6-diazo-5-oxonorleucine; and (4) general
metabolic suppression by lowered temperature.
The only treatment found effective in preventing the mosaicism was lowered temperature,
from which resulted a sigmoidal curve for per cent mosaics versus incubation temperature. These
results would be consistent with mechanisms iii and/or iv but appear to rule out i and ii.
From the speed with which the antigen markers can be seen to propagate across the cell
membrane, and from the fact that the treatment of parent cells with a variety of metabolic
inhibitors does not inhibit antigen spreading, it appears that the cell surface of heterokaryons
is not a rigid structure, but is ' fluid' enough to allow free ' diffusion' of surface antigens resul-
ting in their intermingling within minutes after the initiation of fusion.
* Present address: Immunochemistry Unit, Princess Margaret Hospital for Children,
Subiaco, W.A. 6008, Australia.
t To whom requests for reprints should be addressed.
21 C EL 7
,320 L. D. Frye and M. Edidin
INTRODUCTION
The surface membranes of animal cells rapidly change shape as the cells move,
form pseudopods, or ingest material from their environment. These rapid changes in
shape suggest that the plasma membrane itself is fluid, rather than rigid in character,
and that at least some of its component macromolecules are free to move relative to
one another within the fluid. We have attempted to demonstrate such freedom of move-
ment using specific antigen markers of 2 unlike cell surfaces. Our experiments show
that marker antigens on surface membranes spread rapidly when unlike cells are fused.
The speed of antigen spread and its insensitivity to a number of metabolic inhibitors
offer some support for the notion of a fluid membrane.
We have approached the problem of mixing unlike, and hence readily differentiated,
cell surface membranes by using Sendai virus to fuse tissue culture cells of mouse and
human origin (Harris & Watkins, 1965). The antigens of the parent cell lines and of
progeny heterokaryons have been visualized by indirect immunofluorescence, using
heteroantiserum to whole human cells, and alloantiserum to mouse histocompatibility
antigens. Both sera were cytotoxic for intact cells in the presence of complement;
alloantisera have previously been shown, by both immunofluorescence and immuno-
ferritin techniques, to bind only to the surface of intact cells (Moller, 1961; Cerot-
tini & Brunner, 1967; Drysdale, Merchant, Shreffler & Parker, 1967; Davis & Silver-
man, 1968; Hammerling et al. 1968).
The surface antigens of heterokaryons between hen erythrocytes and HeLa cells
and between Ehrlich ascites and HeLa cells have previously been studied using mixed
agglutination techniques for antigen localization (Watkins & Grace, 1967; Harris,
Sidebottom, Grace & Bramwell, 1969). In these studies intermixing of surface antigens
was demonstrable within an hour or two of heterokaryon formation. However, the
antigens could not readily be localized, since the marker particles used were several
microns in diameter; also, observations were not made of the earliest time at which
mixing occurred. We have been able to examine heterokaryons within 5 min of their
formation and to show that antigen spread and intermixing occurs within minutes
after membrane fusion. Studies on cells poisoned with a variety of metabolic inhibi-
tors strongly suggest that antigen spread and intermixing requires neither de novo
protein synthesis nor insertion of previously synthesized subunits into surface
membranes.
MATERIALS AND METHODS
Cell lines
cuD. A thymidine-kinase negative (TK-) subline of the mouse ' L' cell, isolated by Dubbs &
Kitt (1964), and kindly provided by Dr H. G. Coon.
VA-2. An 8-azaguanine-resistant subclone, isolated by Weiss, Ephrussi & Scaletta (1968),
obtained from W-18-VA-2, an SV4O-transformed human line which has been free of infective
virus for several years (Ponten, Jensen & Koprowski, 1963).
Sal. An ascites tumour (designated as Sarcoma I), provided by Dr A. A. Kandutsch, The
Jackson Laboratory, and carried in Aff mice; it was used as a convenient source of mouse cells
for absorption of antiglobulin reagents.
, Rapid intermixing of surface antigens 321
Tissue culture
The CIID and VA-2 lines were routinely grown in a modified F-12 medium containing 5 %
foetal calf serum (FCS) (Coon & Weiss, 1969) or in Minimal Essential Medium with 5 % FCS,
5 % Fungizone and 100 units penicillin/ml. The cultures were maintained at 37 °C in a water-
jacketed CO 2 incubator, 98% humidity, 5 % CO 2 .
For experiments or routine passages, cells were harvested with 2-5 % heat-inactivated chicken
serum, 0 2 % trypsin and 0-002 % purified collagenase (Worthington CSL) in Moscona's
(1961) solution, which is referred to as ' C T C ' .
Sensitizing antibodies
Mouse alloantiserum (FAS-2). Preparation: antibodies primarily directed against the H-2 k
histocompatibility antigens were obtained by a series of intraperitoneal injections of CBA/jf
(H-2 k ) mouse mesenteric lymph node and spleen cells into BALBfcJ (H-2 d ) mice (4-recipients:
1 donor). Six injections were given twice weekly, followed by a booster 2 weeks after the last
injection. The animals were bled from the retro-orbital sinus 4 and 5 days post-booster.
Specificity. Reaction with mouse cells (CIID) : Aliquots of 2 5 x io 5 CIID cells were treated in
suspension with o-i ml of two-fold dilutions of FAS-2 from 1/10 to 1/80. The cells were agitated
periodically for 15 min at room temperature at which time they were washed twice in phosphate-
buffered saline (PBS). They were then resuspended in 0-05 ml of fluorescein-labelled rabbit
antimouse IgG, incubated, and washed as above. The cells were then put on to Vaseline-ringed
slides, covered and observed in the fluorescence microscope. Ring reactions as reported by
Moller (1961) were observed with decreasing brightness upon increasing dilutions of the FAS-2.
As maximum brightness was desired, the 1/10 dilution was chosen for all subsequent staining
reactions.
Reaction with human cells (VA-2): No fluorescence was observed when analagous staining
reactions were carried out with human cells.
Reaction with Sendai virus: It was discovered that VA-2 cells pre-treated with Sendai
virus became positive for the FAS-2 sensitization. Normal mouse sera from BALB/cjf, CBAjjf,
DBAI2J and A/Jf strains were also shown to exhibit this anti-Sendai activity. This activity in
FAS-2 was easily absorbed by treating a 1/5 dilution of the antiserum with 333-666 haemag-
glutinating units (HAU)/ml of virus for 30 min at room temperature and overnight at 4 °C.
The absorbing virus was then removed by centrifugation.
Rabbit anti-VA-2 antiserum (RaVA-2) preparation: VA-2 cells were grown in Falcon plastic
Petri dishes, harvested with CTC, and washed 3 times in Hanks's balanced salts solution, BSS
(HEPES-buffered) to remove the foetal calf serum. 2 x io 5 cells were emulsified with Freund's
complete adjuvant (cells : adjuvant = 1 : 2 ) and injected intradermally (flanks and footpads) into
a New Zealand white rabbit. One week later io 6 washed cells were given intradermally (flanks
only; io 5 cells/site). The rabbit was bled from the ear vein 1 and 2 weeks following this second
injection. The sera were heat-inactivated at 56 °C for 30 min, aliquoted and stored at — 30 °C.
Specificity of RaVA-2: Reaction with VA-2: VA-2 cells were seeded on to coverslips
(2-5 x io5/coverslip) and allowed to adhere and spread. The coverslips were then washed with
Hanks's and o-i ml of 2-fold dilutions of the RaVA-2 from 1/2 to 1/256 were added. After
incubation in a moist chamber for 15 min at room temperature, the coverslips were washed
twice in Hanks's BSS and similarly treated with tetramethylrhodamine (TMR)-labelled goat
anti-rabbit IgG (anti-Fc). The cells gave strong fluorescent ring reactions at the lower dilutions
of the sensitizing antibody; the 1/4 dilution was chosen for all subsequent staining reactions.
Reaction with CIID: When analogous staining reactions were carried out with the mouse
cells, a very weak fluorescence was seen in the lower dilutions of the RaVA-2. Consequently,
the serum was routinely absorbed with 5 x 1 0 ci iD/ml of a 1/2 diluted serum (30 min at room
temperature).
Reaction with Sendai virus: The CIID cells, when pre-treated with Sendai virus, gave weak
positive staining with the mouse-absorbed RaVA-2. Therefore, the RaVA-2 was also absorbed
with 333 HAU/ml (30 min at room temperature and overnight at 4 0 C). This doubly absorbed
RaVA-2 then gave a negligible background on the CIID cells.
Printed in Great Britain
THE RAPID INTERMIXING OF CELL SURFACE
ANTIGENS AFTER FORMATION OF MOUSE-
HUMAN HETEROKARYONS
L. D. FRYE* AND M. EDIDINf
Department of Biology, The Johns Hopkins University,
Baltimore, Maryland 21218, U.S.A.
SUMMARY
Cells from established tissue culture lines of mouse {CIID) and human (VA-2) origin were
fused together with Sendai virus, producing heterokaryons bearing both mouse and human
surface antigens which were then followed by the indirect fluorescent antibody method. Within
40 min following fusion, total mixing of both parental antigens occurred in over 90 % of the
heterokaryons.
Mouse H-2 (histocompatibility) and human surface antigens were visualized by successive
treatment of the heterokaryons with a mixture of mouse alloantiserum and rabbit anti-VA-2
antiserum, followed by a mixture of fluorescein-labelled goat anti-mouse IgG and tetramethyl-
rhodamine-labelled goat anti-rabbit IgG(Fc).
The CIID x VA-2 fusions were carried out in suspension and maintained at 37 CC in a shaking
water bath; aliquots were removed at various intervals and stained with the above reagents. The
heterokaryon population was observed to change from an initial one (5-min post-fusion) of
non-mosaics (unmixed cell surfaces of red and green fluorescence) to one of over 90 % mosaics
(total intermixing of the 2fluorochromes)by 40 min after fusion. Mouse-human hybrid lines,
derived from similar fusions, gave fluorescence patterns identical to those of the mosaic
heterokaryons.
Four possible mechanisms would yield such results: (i) a very rapid metabolic turnover of
the antigens; (ii) integration of units into the membrane from a cytoplasmic precursor pool;
(iii) movement, or ' diffusion 'of antigen in the plane of the membrane; or (iv) movement of exist-
ing antigen from one membrane site into the cytoplasm and its emergence at a new position on
the membrane.
In an effort to distinguish among these possibilities, the following inhibitor treatments were
carried out: (1) both short- and long-term (6-h pre-treatment) inhibition of protein synthesis
by puromycin, cycloheximide, and chloramphenicol; (2) short-term inhibition of ATP forma-
tion by dinitrophenol (DNP) and NaF; (3) short- and long-term inhibition of glutamine-
dependent pathways with the glutamine analogue 6-diazo-5-oxonorleucine; and (4) general
metabolic suppression by lowered temperature.
The only treatment found effective in preventing the mosaicism was lowered temperature,
from which resulted a sigmoidal curve for per cent mosaics versus incubation temperature. These
results would be consistent with mechanisms iii and/or iv but appear to rule out i and ii.
From the speed with which the antigen markers can be seen to propagate across the cell
membrane, and from the fact that the treatment of parent cells with a variety of metabolic
inhibitors does not inhibit antigen spreading, it appears that the cell surface of heterokaryons
is not a rigid structure, but is ' fluid' enough to allow free ' diffusion' of surface antigens resul-
ting in their intermingling within minutes after the initiation of fusion.
* Present address: Immunochemistry Unit, Princess Margaret Hospital for Children,
Subiaco, W.A. 6008, Australia.
t To whom requests for reprints should be addressed.
21 C EL 7
,320 L. D. Frye and M. Edidin
INTRODUCTION
The surface membranes of animal cells rapidly change shape as the cells move,
form pseudopods, or ingest material from their environment. These rapid changes in
shape suggest that the plasma membrane itself is fluid, rather than rigid in character,
and that at least some of its component macromolecules are free to move relative to
one another within the fluid. We have attempted to demonstrate such freedom of move-
ment using specific antigen markers of 2 unlike cell surfaces. Our experiments show
that marker antigens on surface membranes spread rapidly when unlike cells are fused.
The speed of antigen spread and its insensitivity to a number of metabolic inhibitors
offer some support for the notion of a fluid membrane.
We have approached the problem of mixing unlike, and hence readily differentiated,
cell surface membranes by using Sendai virus to fuse tissue culture cells of mouse and
human origin (Harris & Watkins, 1965). The antigens of the parent cell lines and of
progeny heterokaryons have been visualized by indirect immunofluorescence, using
heteroantiserum to whole human cells, and alloantiserum to mouse histocompatibility
antigens. Both sera were cytotoxic for intact cells in the presence of complement;
alloantisera have previously been shown, by both immunofluorescence and immuno-
ferritin techniques, to bind only to the surface of intact cells (Moller, 1961; Cerot-
tini & Brunner, 1967; Drysdale, Merchant, Shreffler & Parker, 1967; Davis & Silver-
man, 1968; Hammerling et al. 1968).
The surface antigens of heterokaryons between hen erythrocytes and HeLa cells
and between Ehrlich ascites and HeLa cells have previously been studied using mixed
agglutination techniques for antigen localization (Watkins & Grace, 1967; Harris,
Sidebottom, Grace & Bramwell, 1969). In these studies intermixing of surface antigens
was demonstrable within an hour or two of heterokaryon formation. However, the
antigens could not readily be localized, since the marker particles used were several
microns in diameter; also, observations were not made of the earliest time at which
mixing occurred. We have been able to examine heterokaryons within 5 min of their
formation and to show that antigen spread and intermixing occurs within minutes
after membrane fusion. Studies on cells poisoned with a variety of metabolic inhibi-
tors strongly suggest that antigen spread and intermixing requires neither de novo
protein synthesis nor insertion of previously synthesized subunits into surface
membranes.
MATERIALS AND METHODS
Cell lines
cuD. A thymidine-kinase negative (TK-) subline of the mouse ' L' cell, isolated by Dubbs &
Kitt (1964), and kindly provided by Dr H. G. Coon.
VA-2. An 8-azaguanine-resistant subclone, isolated by Weiss, Ephrussi & Scaletta (1968),
obtained from W-18-VA-2, an SV4O-transformed human line which has been free of infective
virus for several years (Ponten, Jensen & Koprowski, 1963).
Sal. An ascites tumour (designated as Sarcoma I), provided by Dr A. A. Kandutsch, The
Jackson Laboratory, and carried in Aff mice; it was used as a convenient source of mouse cells
for absorption of antiglobulin reagents.
, Rapid intermixing of surface antigens 321
Tissue culture
The CIID and VA-2 lines were routinely grown in a modified F-12 medium containing 5 %
foetal calf serum (FCS) (Coon & Weiss, 1969) or in Minimal Essential Medium with 5 % FCS,
5 % Fungizone and 100 units penicillin/ml. The cultures were maintained at 37 °C in a water-
jacketed CO 2 incubator, 98% humidity, 5 % CO 2 .
For experiments or routine passages, cells were harvested with 2-5 % heat-inactivated chicken
serum, 0 2 % trypsin and 0-002 % purified collagenase (Worthington CSL) in Moscona's
(1961) solution, which is referred to as ' C T C ' .
Sensitizing antibodies
Mouse alloantiserum (FAS-2). Preparation: antibodies primarily directed against the H-2 k
histocompatibility antigens were obtained by a series of intraperitoneal injections of CBA/jf
(H-2 k ) mouse mesenteric lymph node and spleen cells into BALBfcJ (H-2 d ) mice (4-recipients:
1 donor). Six injections were given twice weekly, followed by a booster 2 weeks after the last
injection. The animals were bled from the retro-orbital sinus 4 and 5 days post-booster.
Specificity. Reaction with mouse cells (CIID) : Aliquots of 2 5 x io 5 CIID cells were treated in
suspension with o-i ml of two-fold dilutions of FAS-2 from 1/10 to 1/80. The cells were agitated
periodically for 15 min at room temperature at which time they were washed twice in phosphate-
buffered saline (PBS). They were then resuspended in 0-05 ml of fluorescein-labelled rabbit
antimouse IgG, incubated, and washed as above. The cells were then put on to Vaseline-ringed
slides, covered and observed in the fluorescence microscope. Ring reactions as reported by
Moller (1961) were observed with decreasing brightness upon increasing dilutions of the FAS-2.
As maximum brightness was desired, the 1/10 dilution was chosen for all subsequent staining
reactions.
Reaction with human cells (VA-2): No fluorescence was observed when analagous staining
reactions were carried out with human cells.
Reaction with Sendai virus: It was discovered that VA-2 cells pre-treated with Sendai
virus became positive for the FAS-2 sensitization. Normal mouse sera from BALB/cjf, CBAjjf,
DBAI2J and A/Jf strains were also shown to exhibit this anti-Sendai activity. This activity in
FAS-2 was easily absorbed by treating a 1/5 dilution of the antiserum with 333-666 haemag-
glutinating units (HAU)/ml of virus for 30 min at room temperature and overnight at 4 °C.
The absorbing virus was then removed by centrifugation.
Rabbit anti-VA-2 antiserum (RaVA-2) preparation: VA-2 cells were grown in Falcon plastic
Petri dishes, harvested with CTC, and washed 3 times in Hanks's balanced salts solution, BSS
(HEPES-buffered) to remove the foetal calf serum. 2 x io 5 cells were emulsified with Freund's
complete adjuvant (cells : adjuvant = 1 : 2 ) and injected intradermally (flanks and footpads) into
a New Zealand white rabbit. One week later io 6 washed cells were given intradermally (flanks
only; io 5 cells/site). The rabbit was bled from the ear vein 1 and 2 weeks following this second
injection. The sera were heat-inactivated at 56 °C for 30 min, aliquoted and stored at — 30 °C.
Specificity of RaVA-2: Reaction with VA-2: VA-2 cells were seeded on to coverslips
(2-5 x io5/coverslip) and allowed to adhere and spread. The coverslips were then washed with
Hanks's and o-i ml of 2-fold dilutions of the RaVA-2 from 1/2 to 1/256 were added. After
incubation in a moist chamber for 15 min at room temperature, the coverslips were washed
twice in Hanks's BSS and similarly treated with tetramethylrhodamine (TMR)-labelled goat
anti-rabbit IgG (anti-Fc). The cells gave strong fluorescent ring reactions at the lower dilutions
of the sensitizing antibody; the 1/4 dilution was chosen for all subsequent staining reactions.
Reaction with CIID: When analogous staining reactions were carried out with the mouse
cells, a very weak fluorescence was seen in the lower dilutions of the RaVA-2. Consequently,
the serum was routinely absorbed with 5 x 1 0 ci iD/ml of a 1/2 diluted serum (30 min at room
temperature).
Reaction with Sendai virus: The CIID cells, when pre-treated with Sendai virus, gave weak
positive staining with the mouse-absorbed RaVA-2. Therefore, the RaVA-2 was also absorbed
with 333 HAU/ml (30 min at room temperature and overnight at 4 0 C). This doubly absorbed
RaVA-2 then gave a negligible background on the CIID cells.
