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Chemically induced fusion of fresh human erythrocytes
Vol. 86, No. 3, 1979
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
BIOCHEMICAL
Pages 920-928
14, 1979
February
CHEMICALLY INDUCED FUSION OF FRESH HUMAN ERYTHROCYTES Richard
F. Baker
and Vijay
K. Kalra*
Department of Microbiology and Biochemistry* University of Southern California School of Medicine Los Angeles, California 90033 Received
January
3, 1979
The fusion of fresh human erythrocytes was shown to be induced by calcium and phosphate ions. Prior treatment of erythrocytes with phosphate ion was a pre-requisite for the calcium-induced fusion. ATP levels in cells incubated with phosphate and calcium decreased 46 fold while cell-associated calcium increased 70 fold during 1 hour of incubation at 37°C as compared to cells which were incubated with calcium in saline. Our results suggest that a phosphate complex formed bridges between adjacent erythrocytes causing agglutination followed by aggregation of membrane proteins leading to proteinfree areas of lipids. Where these protein-free areas are in close contact fusion may occur.
SummY:
INTRODUCTION:The mechanism common to many biological virus
entry
Sendai
into
virus
using
cell
(1) fusion
have renewed
agent
in vitro and for
used Sendai
interest
is of interest
such as mitosis,
several
approach
years During
Such studies
in the mechanism
of fusion
as compared
because
phagocytosis,
was first
virus.
have been described.
of the chemical
fusion
is
fertilization,
carried
out using
most
investigators
the past
ten years
of chemically because
other
induced
of the apparent
to the complexity
of virus-
fusion.
The fusion
fusion
(2-S)
fusion
Fusion
as a tool
fusion
calcium
etc.
as a fusing
agents
induced
events,
cells,
fusing
simplicity
of cell
of human red blood
and phosphate by these
in the presence
agents
of fresh
phate
concomitant
Abbreviations:
ghosts
has been described
is ATP-dependent
of phosphate
the fusion with
ions
cell
ATP-replete
measurements
(6).
and that
and calcium intact
by the combined
did
action
The authors fresh
intact
of
reported cells
that
agglutinated
not
fuse.
We report
human red
cells
by calcium
and phos-
ATP levels,
percent
of calcium
influx,
DIDS; 4,4'-diisothiocyano-2,2'-stilbene phosphate buffered saline.
disulfonic
here
acid;
PBS
0006-291X/79/030920-09$01.00/0 920
Copyright All rights
@ 1979
by Academic Press, Inc. in any form reserved
of reproduction
Vol.
86, No.
BIOCHEMICAL
3, 1979
hemolysis
and fusion
iOn with
the
of cell
index.
external
membrane
BIOPHYSICAL
Our studies
surface
induced
AND
of cell
by calcium
RESEARCH
show that membrane
is
COMMUNICATIONS
interaction
of phosphate
a prerequisite
in the fusion
ion.
MATERIALSANDMETHODS: Blood was drawn in heparin from human volunteers and used on the same day. It was washed 3x in saline (pH 7.0, 300 m0sm) and resuspended in 10-44 mM phosphate buffered saline (11 mM KH2PO4; 60 mM Na2HP04; 147 ti4 NaCl, pH 7.4). Appropriate volumes of 80 mM CaC12 in saline were added to make a final concentration of 2-20 mM and cell volume adjusted to 10% PCV. Alternatively, after 10 minutes in phosphate buffered Saline, the cells were centrifuged and the supernatant was discarded. To the pellet CaC12 (2-20 mM) in saline was added and volume adjusted to a hematocrit of 10%. After 10 minutes at room temperature, the cell suspension was further incubated at 37°C for varying periods of time. ATP content in the cell was measured by the firefly method of Stanley and Williams (7). Calcium was assayed by atomic absorption spectrophotometry (Perkin-Elmer, Model 290) using an air-acetylene flame. Cells were prepared for analysis by digestion with 5 volumes of 10% nitric acid (Ultrex, J. T. Baker). Lanthanum oxide (0.5%) was added to all specimens and standards to inhibit phosphate interference. The measured volumes were corrected for calcium loss resulting from hemolysis during the washing steps. Freeze-fracture was done with a Polaron unit (Polaron Corp., Line LexFreezing of a 25% glycerol suspension of glutaraldehyde-fixed ington, PA). cells was in Freon 22 at -15O'C. Washed cells were pre-treated with 2-10 p&4, 4,4'-diisothiocyano-2, 2'-stilbene disulfonic acid (DIDS) for 15 minutes at 4'C to inhibit transport of phosphate ion (8). Fusion indices (9) were calculated from the relation F.I.=N, / Nt -1, where No= number of cells at time 0, Nt= number of cells after incubation for No and Nt were counted in a Model A Coulter Counter with a 70 pm time t. aperture. Percent hemolysis was measured by hemoglobin absorption on a Klett calorimeter or Cary 14 spectrophotometer at 415 nm. RESULTS: Freshly (PBS,
human red cells
2-20 mM) agglutinated
saline
was added.
bated
at
after
20-30
fusion
of ghosts
phate
37OC for min.
As a check
to the pellet.
varying
periods (Fig.
caused
and the
process,
On vortexing
of cells
(2-20
were
was typically
report
on the
and phos-
of calcium
hemolysis
incu-
observed
a previous
to a mixture
saline
mM) in
the cells
with
agglutination,
or fusion.
Neither
agglutination. of a visible
precipitate
resuspendedin
removed. this
Fusion
In agreement
redcells
supernatant
when calcium
of time.
in causing alone
in phosphate-buffered
at room temperature,
1)
on the importance
to the fusion
centrifuged
10 min.
(6) the addition
or phosphate
suspended
in a few minutes
After
at 37'C
was ineffective
calcium
phate
drawn
mixture
921
Then calcium the cells
of calcium
phosphate in saline immediately
buffer
phoswere
was added agglutinated
Vol. 86, No. 3, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
electron micrographs of human red cells aggregated and Figure 1. Scanning fused by exposure to phosphate ion and calcium. (a) After 30 min. incubation at 37"C, several stages of fusion of red cells exposed to 20 mk4 Ca after pre-treatment of cells with 13,mM phosphate are seen. Single cells appear at lower left and upper right, a probable doublet at lower right and a large polykaron in the center with cells in process of fusion. @I A clump of aggregated and fusing cells show emerging vesicles. The cells of dehydrated (a) and (b) were fixed after 30 min. at 37°C by 2% glutaraldehyde, in ethanol, and critical point dried in liquid carbon dioxide. Sputter coating with gold-palladium preceded the examination of the cells in an AMR-1000 scanning microscope ( X5000).
and then
on incubation
hemolysis.
Thus
or no role
the visible
was that
higher
than
under
tended
to obscure
pellet
of cells
was seen,
detail.
sample isotonic
increased
of calcium
under
the phase
a precipitate hand,
if
between
and fusion
indices
20 mM calcium-saline
washing
was suspended
incubation
of freshly
drawn
in 44 mk4 phosphate
After
to phosphate
cell-cell
are
shown in Fig.
2 for
blood
in isotonic saline,
be-
bridging. percent
exposure
to a
no agglutination
the cells
the two pellets
922
was added
ATP levels,
buffered
centrifugation,
clumps
time,
after
of
was much
the cell
phosphate
little
method
microscope
solution,
to achieve
andreduced
plays
of this
around
of exposing
in order
efficiency
phosphate
One advantage
On the other
of calcium
at 37°C in
saline.
where
the necessity
calcium
thorough
with
spun down from a calcium-saline
The relationships
After
visibility
conditions
the addition
incubated
fused
and fusion.
cell
indicating
cell-associated
they
precipitate
in agglutination
preparation
fore
at 37'C
hemolysis, red cells
to 44 mM phosphate. saline, another
one sample
were resuspended
in
Vol. 86, No. 3, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
lncuba tion Time (mid Figure 2. Effect of incubation time on ATP levels, hemolysis, cell-associated calcium and fusion indices for red cells exposed to calcium-saline containing 44 mM phosphate. Six x lml samples of saline-washed red blood cells were prepared at 10% PCV; four were suspended in 44 mM phosphate-buffered saline pH 7.4, and two samples in 300 mOs saline. All tubes were centrifuged at 1610 g for 5 min., following which the supernatant was withdrawn. One ml of 20 mM CaC12, buffered with 12 mM Hepes to pH 7.4 and made isotonic with NaCl, was added to each pellet and the tubes were vortexed before incubation at 37°C. 0.1 ml of At times 0, 10, 30, and 60 min. and after vortexing, suspension was removed for ATP assay (7). 20 ~1 of suspension was removed and diluted 50,000 x with Isoton for cell counts (Coulter Model A with 70 pm aperture). The remaining suspension in each tube was centrifuged at 2440 g. for 10 min. at 4°C and the supernatant removed for measurement of hemoglobin absorption to give percent hemolysis. The pellet of intact and fused cells and ghosts was washed (10 min. X 2445 g.) 3X in 14 ml of isotonic saline at 4"C, and prepared for calcium assay as described in Materials and Methods. to a 10% PCV in buffered were
incubated
levels
in the
cells
while
cubated
saline
associated saline-calcium hour
containing
at 37°C and aliquots
in 1 hour, in
saline
incubated
cell-associated plus
calcium
suspension,
removed
with
phosphate calcium
20 ti4 calcium,
showed
20 mM calcium
no change
at 0, 10,
in 1 hr.
but was 60% complete
of incubation.
923
decreased
70 fold.
ATP decreased
only
Hemolysis
Both
30 and 60 min.
and calcium
increased
chloride.
ATP 46 fold
In cells 2 fold,
in-
while
was minimal
in phosphate-calcium
tubes
cell-
in the after
1
Vol.
86,
No.
3, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Figure 3. (a) Transmission electron micrograph of early stage of cell fusion. Calcium chloride (final concentration 20 mFI), was added to a suspension (10% PCV) of red cells in 13 mM phosphate buffer. After 20 min. at 37°C the cells were fixed in 2% buffered glutaraldehyde for 30 min. followed by 2% buffered osmium tetroxide for 1 hour. Dehydration in ethanol and embedding in Epon 812 preceded the cutting of thin sections on an LKB Ultratome. Sections were stained with uranyl acetate and lead citrate. A vesicle originating from the lower cell is invaginating into the cell at top. Partial hemolysis is seen in both cells. (b) As in Fig. 3a except for an incubation time of 1 hour. Free intracellular vesicles which presumably originated in a contacting cell are seen. (a) X88,000 (b) X33,600
Entry
into
the red cell
postulated
as one of the
that
of phosphate
entry
fusion
by phosphate
ghost
steps into
of both
in fusion the
of anion
and calcium.
fusion.
Controls
enter
the cells
phosphate
transport established and yet
or calcium
by DIDS treatment. initiated
before
However, hemolysis
of fusion Freeze-fracture
common steps
in fusion
15 minutes under
during
studies
treated
nM),
that
fusion a later
mechanism.
Fig.
much of their
924
is
tested
for did
The entry not
out been
of phosphate
3A shows an early
an aggregation
that
not
of
ruled
had already entry
DIDS
a known in-
hemoglobin.
have established
agents
for with
phosphate
is of course
so that
(5,11,12)
by various
2-10
conditions
hemolysis
was complete
containing
were
was unimpaired.
seems likely
probable
at 4°C and then
these
has been
may be not necessary
acid;
on the fusion
in cells
cell
disulfonic
efficiency
it
It now appears
in saline
that
fusion
red
and phosphate
Red cells
for
phosphate
may have had no bearing stage
(8),
(10).
intact
(4,4'-diisothiocyano-2,2'-stilbene hibitor
calcium
one of the in the
fusing
Vol.
86,
No.
3, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Figure 4. Aggregation of membrane-associated protein and protein-free vesiculation are seen in this replica of freeze-fractured cells which had been incubated at 37°C in 13 mM phosphate-20 mM calcium. After 20 min. incubation the suspension was fixed in 2% buffered glutataldehyde for 30 min., then washed 3X in 15 volumes of distilled water, before resuspending in 25% glycerol-water. Aliquots (2 Ill) were frozen in Freon 22 at -150°C. Fracture was by the split tube method at -1lO'C in a Polaron freeze-etch unit. A carbon-platinum replica of the fracture surface was examined in a Philips 300 transmission electron microscope (X 53800).
membranes observed It
of membrane-associated for
fusion
of intact
has been reported
protein-free spectrin
vesiculation (13)
the formation phosphate.
that
proteins red cells
or by phase
separation
of MAP-free
vesicles
Such vesicles
calcium
cell
We confirm
by phosphate
intracellular in red
(MAP).
and calcium
causes
ghosts,
perhaps
of lipid
(14,15).
from red
may exvaginate
into
925
cells
that
this
is
(Fig.
4).
MAP aggregation by interaction
treated
the medium,
Fig. with
and with
4 illustrates calcium
or as in Fig.
and 3A
Vol.
86,
No.
3, 1979
BIOCHEMICAL
Table t (min)
I Partitioning
RESEARCH
COMMUNICATIONS
red cells
ATP in the Supernatant %
100
ATP Utilized %
0
0
10
68.1
7.9
24
30
52.8
8.2
39
60
32.5
5.5
62
A suspension
of red cells
buffered
saline,
pH 7.4)
in saline
of this
suspension
suspensions
following
seen in Fig. cellular
four aliquots
at 37 C for 0, 10, 30 and 60 min.
After
centrifugation
neighbor
3B these
by subtraction at a point
at 1610 X g. for 5 min.,
vesicles
of supernatant where
at a later
The
and 0.1 ml removed for
was removed for measurement of supernatant
were obtained
a close
with 12 mkl Hepes,
10 min. at room temperature,
at these time intervals
ATP (7).
0.1 ml of supernatant
bud into
After
were incubated
were vortexed
ATP levels
(10% PCV) was washed in 13 til phosphate-
which 27 IN CaC12 (buffered
was added.
measurement of total
fusion stage
ATP.
Pkllet
ATP from total
is being
ATP.
initiated.
of fusion
will
As
appear
as intra-
vesicles. In order
hemolysis, natant
to distinguish
as a function
After
ghosts
1 hour
while
only
of incubation ATP (38%), 5.5% is
the ATP was consumed calcium
into
calcium
pump is activated If
the cell
before
this
a metabolic
in the complete
of incubation
was nearly
experiment,
on ATP.
between
ATP was measured
Of the remaining
this
BIOPHYSICAL
of ATP in fusing
ATP in the pellet %
0
I.
AND
time.
fusion Some
about found
typical
hemolysis. for
by intracellular the disparity
super-
shown in Table
intact
cells
hemolysis,
it
follows
that
is probable
that
in ATP levels
calcium
and functionally
the ATP drop
and in
most of
the entry
the drop
between
926
are
Since
1 hour It
data
with
in the supernatant. after
and in the
by
ATP had been utilized.
32% is associated
complete
of ATP and a loss
mixture
62% of the original
was responsible
is true,
loss
of
since
in this
the
dependent experi-
Vol.
86,
No.
3, 1979
ment and that cium levels let
BIOCHEMICAL
seen in Fig.
cells
DIscussIolv: human red
with
cells
report
phate
ion with
occurs, (6).
occur
diated
The levels
requires
since did
were
with
efficiency channel
suggesting
with
may not be necessary
content
membrane-associated
protein
protein
crosslinking
tein.
Recent
causes
an increase
brane
More over,
(16).
This
of Alan
may lead
and Mitchell
in the membrane
of removal
of bulky
The mechanism different
than
pre-treatment
that
charged
facilitated
during
the of
aggregates
the
of calcium
results
ion
in membrane
of membrane
have shown that
pro-
calcium
of 1,2-diacylglycerol
as a result
They suggested and diphosphate
that
the
from the mem-
process
as a result
of
groups. induced
by calcium
proposed
by Alan
and Mitchell
with
which
the fusion
me-
has been
levels
to aggregation
of fusion
of cells
by calcium.
phosphate
did
process.
Calcium
presumably
phosphodiesterase.
of phosphatidylinositol of erythrocytes
content
In ad-
The consequences
increased
(17)
the
transport
by 70 fold
fold.
transglutaminase
of membrane-bound
surface
induced
(13).
did
cal-
inhibitor,
the fusion
at 37 C.
which
with
mixture.
phosphate for
several
complex
of intrinsic
studies
are
cells
transport
increased
and phosphate
the spectrin-actin
an activation
calcium
calcium
in calcium
causes
that
of phos-
Neither
phosphate
DIDS, an anion
to a pre-
association
in fusion.
to a calcium
ATP-replete
contrary
of red blood
not result
added
shown to affect
hydrolysis
pre-treatment
the amount
of fresh
ion,
prior
the pel-
much smaller.
fusion
process
of cell-associated
such an increase
shows that
The fusion
of cells
period
of activation
here
cal-
with
since
is normally
and phosphate
the fusion
incubation
presented
by phosphate
by an anion
hemolysis
COMMUNICATIONS
in intracellular
of note,
calcium
pre-treatment
RESEARCH
associated
using
when cells
not affect
is worthy
after
the membrane
followed
dition,
cells
The data
vious
fusion
The ATP still
(32% of original)
of ATP associated
BIOPHYSICAL
2 may be due to differences
in the two experiments.
of fused
cium ion
AND
phosphate
The formation
ion
and phosphate
is required
of 1,2-diacylglycerol
927
(18),
since for
appears in our
the fusion may occur
to be studies process
in this
Vol.
86, No.
chemically
induced
process. bridge
It between
permeability
fusion
appears
is
but
that
adjacent
of the bilayer
finally
BIOCHEMICAL
3, 1979
hemolysis.
to be counteracted
may not
erythrocytes,
and water, enters
by the calcium
With accumulation
of intracellular
tein
occurs,
in protein-free
are
in close
juxtaposition
RESEARCH
COMMUNICATIONS
event
in the fusion
be the primary
causing
by an unknown
Calcium
resulting
BIOPHYSICAL
a phosphate-calcium-phosphate
reduced
to cations
it
AND
agglutination.
mechanism
an increase the
cell
as a result
leading
at a rate
calcium
aggregation
fragility is too
and fast
of ATP falls
rapidly.
of membrane
pro-
Where such areas
of lipids.
of bridging,
a
to an increased
which
the level
forms
The stability
in osmotic
pump and thus
areas
complex
fusion
may be initiated.
ACKNOWLEDGMENTS: This work was supported by grants from National The excellent technical assistance of Mrs. of Health (HL-15162). is gratefully acknowledged.
Institute Linda Clark
REFERENCES: 1. 2. 3. 4. 5. 6. 7. a. 9. 10. 11. 12. 13. 14. 15. 16. 17.
Harris, H. and Watkins, J. F., Nature, Lond., 205, 640-646 (1965) Ahkong, Q, F., Fisher, D., Tampion, W. and LucxJ. A., Biochem. J *, 136, 147-155 (1973) Papahadjopoulos, D., Poste, G. and Schaeffer, B. E., Biochim. Biophys. Acta 323, 23-42 (1973) Ahkong, Q. F., Howell, J. I., Lucy, J. A., Safwat, F., Davey, M. R. and Cocking, E. C., Nature, E, 66-67 (197.5) Ahkong, Q. F., Fisher, D., Tampion, W. and Lucy, J. A., Nature, --253, 194-195 (1975) Zakai, N., Kulka, R. G. and Loyter, A., Nature, 263, 696-699 (1976) Stanley, P. E. and Williams, S. G., Analytical Biochem. 2, 381-392 (1969) Cabantchik, 2. I. and Rothstein, A., J. Memb. Biol. 15, 207-226 (1974) Okada, Y. and Tadokoro, J., Ex. Cell. Res., 2, 108-118 (1962) Zakai, N., Kulka, R. G. and Loyter, A., Proc. Nat. Acad. Sci., 2, 2417-2421 (1977) 1004-1012 (1973) Bachi, T., Aguet, M. and Howe, C., J. Virol.,E, Vos, J., Ahkcng, Q. F., Botham, G. M., Qurik, S. D. and Lucy, J. A., Biochem. J., 158, 651-653 (1976) D. M. and Branton, D., Biochim. Biophys. Acta, Elgsaeter, A., Shotton, 426, 101-122 (1976) James, R. and Branton, D,, Biochim. Biophys. Acta, 323, 378-390 (1973) Jacobson, K. and Papahadjopoulos, D., Biochem., 14, 152-161 (1975) Lorand, L., Weissman, L. B., Epel, D. L. and Lorand, J. B., Proc. Natl. Acad. Sci. 73, 4479-4481 (1976) Allan, D. and Mitchell, R. H., Biochim. Biophys. Acta 508, 277-286 (1978)
928
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
BIOCHEMICAL
Pages 920-928
14, 1979
February
CHEMICALLY INDUCED FUSION OF FRESH HUMAN ERYTHROCYTES Richard
F. Baker
and Vijay
K. Kalra*
Department of Microbiology and Biochemistry* University of Southern California School of Medicine Los Angeles, California 90033 Received
January
3, 1979
The fusion of fresh human erythrocytes was shown to be induced by calcium and phosphate ions. Prior treatment of erythrocytes with phosphate ion was a pre-requisite for the calcium-induced fusion. ATP levels in cells incubated with phosphate and calcium decreased 46 fold while cell-associated calcium increased 70 fold during 1 hour of incubation at 37°C as compared to cells which were incubated with calcium in saline. Our results suggest that a phosphate complex formed bridges between adjacent erythrocytes causing agglutination followed by aggregation of membrane proteins leading to proteinfree areas of lipids. Where these protein-free areas are in close contact fusion may occur.
SummY:
INTRODUCTION:The mechanism common to many biological virus
entry
Sendai
into
virus
using
cell
(1) fusion
have renewed
agent
in vitro and for
used Sendai
interest
is of interest
such as mitosis,
several
approach
years During
Such studies
in the mechanism
of fusion
as compared
because
phagocytosis,
was first
virus.
have been described.
of the chemical
fusion
is
fertilization,
carried
out using
most
investigators
the past
ten years
of chemically because
other
induced
of the apparent
to the complexity
of virus-
fusion.
The fusion
fusion
(2-S)
fusion
Fusion
as a tool
fusion
calcium
etc.
as a fusing
agents
induced
events,
cells,
fusing
simplicity
of cell
of human red blood
and phosphate by these
in the presence
agents
of fresh
phate
concomitant
Abbreviations:
ghosts
has been described
is ATP-dependent
of phosphate
the fusion with
ions
cell
ATP-replete
measurements
(6).
and that
and calcium intact
by the combined
did
action
The authors fresh
intact
of
reported cells
that
agglutinated
not
fuse.
We report
human red
cells
by calcium
and phos-
ATP levels,
percent
of calcium
influx,
DIDS; 4,4'-diisothiocyano-2,2'-stilbene phosphate buffered saline.
disulfonic
here
acid;
PBS
0006-291X/79/030920-09$01.00/0 920
Copyright All rights
@ 1979
by Academic Press, Inc. in any form reserved
of reproduction
Vol.
86, No.
BIOCHEMICAL
3, 1979
hemolysis
and fusion
iOn with
the
of cell
index.
external
membrane
BIOPHYSICAL
Our studies
surface
induced
AND
of cell
by calcium
RESEARCH
show that membrane
is
COMMUNICATIONS
interaction
of phosphate
a prerequisite
in the fusion
ion.
MATERIALSANDMETHODS: Blood was drawn in heparin from human volunteers and used on the same day. It was washed 3x in saline (pH 7.0, 300 m0sm) and resuspended in 10-44 mM phosphate buffered saline (11 mM KH2PO4; 60 mM Na2HP04; 147 ti4 NaCl, pH 7.4). Appropriate volumes of 80 mM CaC12 in saline were added to make a final concentration of 2-20 mM and cell volume adjusted to 10% PCV. Alternatively, after 10 minutes in phosphate buffered Saline, the cells were centrifuged and the supernatant was discarded. To the pellet CaC12 (2-20 mM) in saline was added and volume adjusted to a hematocrit of 10%. After 10 minutes at room temperature, the cell suspension was further incubated at 37°C for varying periods of time. ATP content in the cell was measured by the firefly method of Stanley and Williams (7). Calcium was assayed by atomic absorption spectrophotometry (Perkin-Elmer, Model 290) using an air-acetylene flame. Cells were prepared for analysis by digestion with 5 volumes of 10% nitric acid (Ultrex, J. T. Baker). Lanthanum oxide (0.5%) was added to all specimens and standards to inhibit phosphate interference. The measured volumes were corrected for calcium loss resulting from hemolysis during the washing steps. Freeze-fracture was done with a Polaron unit (Polaron Corp., Line LexFreezing of a 25% glycerol suspension of glutaraldehyde-fixed ington, PA). cells was in Freon 22 at -15O'C. Washed cells were pre-treated with 2-10 p&4, 4,4'-diisothiocyano-2, 2'-stilbene disulfonic acid (DIDS) for 15 minutes at 4'C to inhibit transport of phosphate ion (8). Fusion indices (9) were calculated from the relation F.I.=N, / Nt -1, where No= number of cells at time 0, Nt= number of cells after incubation for No and Nt were counted in a Model A Coulter Counter with a 70 pm time t. aperture. Percent hemolysis was measured by hemoglobin absorption on a Klett calorimeter or Cary 14 spectrophotometer at 415 nm. RESULTS: Freshly (PBS,
human red cells
2-20 mM) agglutinated
saline
was added.
bated
at
after
20-30
fusion
of ghosts
phate
37OC for min.
As a check
to the pellet.
varying
periods (Fig.
caused
and the
process,
On vortexing
of cells
(2-20
were
was typically
report
on the
and phos-
of calcium
hemolysis
incu-
observed
a previous
to a mixture
saline
mM) in
the cells
with
agglutination,
or fusion.
Neither
agglutination. of a visible
precipitate
resuspendedin
removed. this
Fusion
In agreement
redcells
supernatant
when calcium
of time.
in causing alone
in phosphate-buffered
at room temperature,
1)
on the importance
to the fusion
centrifuged
10 min.
(6) the addition
or phosphate
suspended
in a few minutes
After
at 37'C
was ineffective
calcium
phate
drawn
mixture
921
Then calcium the cells
of calcium
phosphate in saline immediately
buffer
phoswere
was added agglutinated
Vol. 86, No. 3, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
electron micrographs of human red cells aggregated and Figure 1. Scanning fused by exposure to phosphate ion and calcium. (a) After 30 min. incubation at 37"C, several stages of fusion of red cells exposed to 20 mk4 Ca after pre-treatment of cells with 13,mM phosphate are seen. Single cells appear at lower left and upper right, a probable doublet at lower right and a large polykaron in the center with cells in process of fusion. @I A clump of aggregated and fusing cells show emerging vesicles. The cells of dehydrated (a) and (b) were fixed after 30 min. at 37°C by 2% glutaraldehyde, in ethanol, and critical point dried in liquid carbon dioxide. Sputter coating with gold-palladium preceded the examination of the cells in an AMR-1000 scanning microscope ( X5000).
and then
on incubation
hemolysis.
Thus
or no role
the visible
was that
higher
than
under
tended
to obscure
pellet
of cells
was seen,
detail.
sample isotonic
increased
of calcium
under
the phase
a precipitate hand,
if
between
and fusion
indices
20 mM calcium-saline
washing
was suspended
incubation
of freshly
drawn
in 44 mk4 phosphate
After
to phosphate
cell-cell
are
shown in Fig.
2 for
blood
in isotonic saline,
be-
bridging. percent
exposure
to a
no agglutination
the cells
the two pellets
922
was added
ATP levels,
buffered
centrifugation,
clumps
time,
after
of
was much
the cell
phosphate
little
method
microscope
solution,
to achieve
andreduced
plays
of this
around
of exposing
in order
efficiency
phosphate
One advantage
On the other
of calcium
at 37°C in
saline.
where
the necessity
calcium
thorough
with
spun down from a calcium-saline
The relationships
After
visibility
conditions
the addition
incubated
fused
and fusion.
cell
indicating
cell-associated
they
precipitate
in agglutination
preparation
fore
at 37'C
hemolysis, red cells
to 44 mM phosphate. saline, another
one sample
were resuspended
in
Vol. 86, No. 3, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
lncuba tion Time (mid Figure 2. Effect of incubation time on ATP levels, hemolysis, cell-associated calcium and fusion indices for red cells exposed to calcium-saline containing 44 mM phosphate. Six x lml samples of saline-washed red blood cells were prepared at 10% PCV; four were suspended in 44 mM phosphate-buffered saline pH 7.4, and two samples in 300 mOs saline. All tubes were centrifuged at 1610 g for 5 min., following which the supernatant was withdrawn. One ml of 20 mM CaC12, buffered with 12 mM Hepes to pH 7.4 and made isotonic with NaCl, was added to each pellet and the tubes were vortexed before incubation at 37°C. 0.1 ml of At times 0, 10, 30, and 60 min. and after vortexing, suspension was removed for ATP assay (7). 20 ~1 of suspension was removed and diluted 50,000 x with Isoton for cell counts (Coulter Model A with 70 pm aperture). The remaining suspension in each tube was centrifuged at 2440 g. for 10 min. at 4°C and the supernatant removed for measurement of hemoglobin absorption to give percent hemolysis. The pellet of intact and fused cells and ghosts was washed (10 min. X 2445 g.) 3X in 14 ml of isotonic saline at 4"C, and prepared for calcium assay as described in Materials and Methods. to a 10% PCV in buffered were
incubated
levels
in the
cells
while
cubated
saline
associated saline-calcium hour
containing
at 37°C and aliquots
in 1 hour, in
saline
incubated
cell-associated plus
calcium
suspension,
removed
with
phosphate calcium
20 ti4 calcium,
showed
20 mM calcium
no change
at 0, 10,
in 1 hr.
but was 60% complete
of incubation.
923
decreased
70 fold.
ATP decreased
only
Hemolysis
Both
30 and 60 min.
and calcium
increased
chloride.
ATP 46 fold
In cells 2 fold,
in-
while
was minimal
in phosphate-calcium
tubes
cell-
in the after
1
Vol.
86,
No.
3, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Figure 3. (a) Transmission electron micrograph of early stage of cell fusion. Calcium chloride (final concentration 20 mFI), was added to a suspension (10% PCV) of red cells in 13 mM phosphate buffer. After 20 min. at 37°C the cells were fixed in 2% buffered glutaraldehyde for 30 min. followed by 2% buffered osmium tetroxide for 1 hour. Dehydration in ethanol and embedding in Epon 812 preceded the cutting of thin sections on an LKB Ultratome. Sections were stained with uranyl acetate and lead citrate. A vesicle originating from the lower cell is invaginating into the cell at top. Partial hemolysis is seen in both cells. (b) As in Fig. 3a except for an incubation time of 1 hour. Free intracellular vesicles which presumably originated in a contacting cell are seen. (a) X88,000 (b) X33,600
Entry
into
the red cell
postulated
as one of the
that
of phosphate
entry
fusion
by phosphate
ghost
steps into
of both
in fusion the
of anion
and calcium.
fusion.
Controls
enter
the cells
phosphate
transport established and yet
or calcium
by DIDS treatment. initiated
before
However, hemolysis
of fusion Freeze-fracture
common steps
in fusion
15 minutes under
during
studies
treated
nM),
that
fusion a later
mechanism.
Fig.
much of their
924
is
tested
for did
The entry not
out been
of phosphate
3A shows an early
an aggregation
that
not
of
ruled
had already entry
DIDS
a known in-
hemoglobin.
have established
agents
for with
phosphate
is of course
so that
(5,11,12)
by various
2-10
conditions
hemolysis
was complete
containing
were
was unimpaired.
seems likely
probable
at 4°C and then
these
has been
may be not necessary
acid;
on the fusion
in cells
cell
disulfonic
efficiency
it
It now appears
in saline
that
fusion
red
and phosphate
Red cells
for
phosphate
may have had no bearing stage
(8),
(10).
intact
(4,4'-diisothiocyano-2,2'-stilbene hibitor
calcium
one of the in the
fusing
Vol.
86,
No.
3, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Figure 4. Aggregation of membrane-associated protein and protein-free vesiculation are seen in this replica of freeze-fractured cells which had been incubated at 37°C in 13 mM phosphate-20 mM calcium. After 20 min. incubation the suspension was fixed in 2% buffered glutataldehyde for 30 min., then washed 3X in 15 volumes of distilled water, before resuspending in 25% glycerol-water. Aliquots (2 Ill) were frozen in Freon 22 at -150°C. Fracture was by the split tube method at -1lO'C in a Polaron freeze-etch unit. A carbon-platinum replica of the fracture surface was examined in a Philips 300 transmission electron microscope (X 53800).
membranes observed It
of membrane-associated for
fusion
of intact
has been reported
protein-free spectrin
vesiculation (13)
the formation phosphate.
that
proteins red cells
or by phase
separation
of MAP-free
vesicles
Such vesicles
calcium
cell
We confirm
by phosphate
intracellular in red
(MAP).
and calcium
causes
ghosts,
perhaps
of lipid
(14,15).
from red
may exvaginate
into
925
cells
that
this
is
(Fig.
4).
MAP aggregation by interaction
treated
the medium,
Fig. with
and with
4 illustrates calcium
or as in Fig.
and 3A
Vol.
86,
No.
3, 1979
BIOCHEMICAL
Table t (min)
I Partitioning
RESEARCH
COMMUNICATIONS
red cells
ATP in the Supernatant %
100
ATP Utilized %
0
0
10
68.1
7.9
24
30
52.8
8.2
39
60
32.5
5.5
62
A suspension
of red cells
buffered
saline,
pH 7.4)
in saline
of this
suspension
suspensions
following
seen in Fig. cellular
four aliquots
at 37 C for 0, 10, 30 and 60 min.
After
centrifugation
neighbor
3B these
by subtraction at a point
at 1610 X g. for 5 min.,
vesicles
of supernatant where
at a later
The
and 0.1 ml removed for
was removed for measurement of supernatant
were obtained
a close
with 12 mkl Hepes,
10 min. at room temperature,
at these time intervals
ATP (7).
0.1 ml of supernatant
bud into
After
were incubated
were vortexed
ATP levels
(10% PCV) was washed in 13 til phosphate-
which 27 IN CaC12 (buffered
was added.
measurement of total
fusion stage
ATP.
Pkllet
ATP from total
is being
ATP.
initiated.
of fusion
will
As
appear
as intra-
vesicles. In order
hemolysis, natant
to distinguish
as a function
After
ghosts
1 hour
while
only
of incubation ATP (38%), 5.5% is
the ATP was consumed calcium
into
calcium
pump is activated If
the cell
before
this
a metabolic
in the complete
of incubation
was nearly
experiment,
on ATP.
between
ATP was measured
Of the remaining
this
BIOPHYSICAL
of ATP in fusing
ATP in the pellet %
0
I.
AND
time.
fusion Some
about found
typical
hemolysis. for
by intracellular the disparity
super-
shown in Table
intact
cells
hemolysis,
it
follows
that
is probable
that
in ATP levels
calcium
and functionally
the ATP drop
and in
most of
the entry
the drop
between
926
are
Since
1 hour It
data
with
in the supernatant. after
and in the
by
ATP had been utilized.
32% is associated
complete
of ATP and a loss
mixture
62% of the original
was responsible
is true,
loss
of
since
in this
the
dependent experi-
Vol.
86,
No.
3, 1979
ment and that cium levels let
BIOCHEMICAL
seen in Fig.
cells
DIscussIolv: human red
with
cells
report
phate
ion with
occurs, (6).
occur
diated
The levels
requires
since did
were
with
efficiency channel
suggesting
with
may not be necessary
content
membrane-associated
protein
protein
crosslinking
tein.
Recent
causes
an increase
brane
More over,
(16).
This
of Alan
may lead
and Mitchell
in the membrane
of removal
of bulky
The mechanism different
than
pre-treatment
that
charged
facilitated
during
the of
aggregates
the
of calcium
results
ion
in membrane
of membrane
have shown that
pro-
calcium
of 1,2-diacylglycerol
as a result
They suggested and diphosphate
that
the
from the mem-
process
as a result
of
groups. induced
by calcium
proposed
by Alan
and Mitchell
with
which
the fusion
me-
has been
levels
to aggregation
of fusion
of cells
by calcium.
phosphate
did
process.
Calcium
presumably
phosphodiesterase.
of phosphatidylinositol of erythrocytes
content
In ad-
The consequences
increased
(17)
the
transport
by 70 fold
fold.
transglutaminase
of membrane-bound
surface
induced
(13).
did
cal-
inhibitor,
the fusion
at 37 C.
which
with
mixture.
phosphate for
several
complex
of intrinsic
studies
are
cells
transport
increased
and phosphate
the spectrin-actin
an activation
calcium
calcium
in calcium
causes
that
of phos-
Neither
phosphate
DIDS, an anion
to a pre-
association
in fusion.
to a calcium
ATP-replete
contrary
of red blood
not result
added
shown to affect
hydrolysis
pre-treatment
the amount
of fresh
ion,
prior
the pel-
much smaller.
fusion
process
of cell-associated
such an increase
shows that
The fusion
of cells
period
of activation
here
cal-
with
since
is normally
and phosphate
the fusion
incubation
presented
by phosphate
by an anion
hemolysis
COMMUNICATIONS
in intracellular
of note,
calcium
pre-treatment
RESEARCH
associated
using
when cells
not affect
is worthy
after
the membrane
followed
dition,
cells
The data
vious
fusion
The ATP still
(32% of original)
of ATP associated
BIOPHYSICAL
2 may be due to differences
in the two experiments.
of fused
cium ion
AND
phosphate
The formation
ion
and phosphate
is required
of 1,2-diacylglycerol
927
(18),
since for
appears in our
the fusion may occur
to be studies process
in this
Vol.
86, No.
chemically
induced
process. bridge
It between
permeability
fusion
appears
is
but
that
adjacent
of the bilayer
finally
BIOCHEMICAL
3, 1979
hemolysis.
to be counteracted
may not
erythrocytes,
and water, enters
by the calcium
With accumulation
of intracellular
tein
occurs,
in protein-free
are
in close
juxtaposition
RESEARCH
COMMUNICATIONS
event
in the fusion
be the primary
causing
by an unknown
Calcium
resulting
BIOPHYSICAL
a phosphate-calcium-phosphate
reduced
to cations
it
AND
agglutination.
mechanism
an increase the
cell
as a result
leading
at a rate
calcium
aggregation
fragility is too
and fast
of ATP falls
rapidly.
of membrane
pro-
Where such areas
of lipids.
of bridging,
a
to an increased
which
the level
forms
The stability
in osmotic
pump and thus
areas
complex
fusion
may be initiated.
ACKNOWLEDGMENTS: This work was supported by grants from National The excellent technical assistance of Mrs. of Health (HL-15162). is gratefully acknowledged.
Institute Linda Clark
REFERENCES: 1. 2. 3. 4. 5. 6. 7. a. 9. 10. 11. 12. 13. 14. 15. 16. 17.
Harris, H. and Watkins, J. F., Nature, Lond., 205, 640-646 (1965) Ahkong, Q, F., Fisher, D., Tampion, W. and LucxJ. A., Biochem. J *, 136, 147-155 (1973) Papahadjopoulos, D., Poste, G. and Schaeffer, B. E., Biochim. Biophys. Acta 323, 23-42 (1973) Ahkong, Q. F., Howell, J. I., Lucy, J. A., Safwat, F., Davey, M. R. and Cocking, E. C., Nature, E, 66-67 (197.5) Ahkong, Q. F., Fisher, D., Tampion, W. and Lucy, J. A., Nature, --253, 194-195 (1975) Zakai, N., Kulka, R. G. and Loyter, A., Nature, 263, 696-699 (1976) Stanley, P. E. and Williams, S. G., Analytical Biochem. 2, 381-392 (1969) Cabantchik, 2. I. and Rothstein, A., J. Memb. Biol. 15, 207-226 (1974) Okada, Y. and Tadokoro, J., Ex. Cell. Res., 2, 108-118 (1962) Zakai, N., Kulka, R. G. and Loyter, A., Proc. Nat. Acad. Sci., 2, 2417-2421 (1977) 1004-1012 (1973) Bachi, T., Aguet, M. and Howe, C., J. Virol.,E, Vos, J., Ahkcng, Q. F., Botham, G. M., Qurik, S. D. and Lucy, J. A., Biochem. J., 158, 651-653 (1976) D. M. and Branton, D., Biochim. Biophys. Acta, Elgsaeter, A., Shotton, 426, 101-122 (1976) James, R. and Branton, D,, Biochim. Biophys. Acta, 323, 378-390 (1973) Jacobson, K. and Papahadjopoulos, D., Biochem., 14, 152-161 (1975) Lorand, L., Weissman, L. B., Epel, D. L. and Lorand, J. B., Proc. Natl. Acad. Sci. 73, 4479-4481 (1976) Allan, D. and Mitchell, R. H., Biochim. Biophys. Acta 508, 277-286 (1978)
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