0022-
1554/79/2712-1610$02.00/0
THE
JOURNAL
Copyright
©
OF HISTOCHEMISTRY
by The
1979
Cellular
AND
Histochemical
Vol.
CYTOCHEMISTRY
Society,
Distribution of High and on the Plasma Membrane MARTHA
Departments
ofBiological
FELLER,
Chemistry
Received
for
No. 12, pp. 1610-1617, 1979 Printedin U.S.A.
27,
Inc.
(M.F.,
publication
Low Affinity of Normal
RANDAL E.G.)
MORRIS,
and Microbiology, Cincinnati, Ohio
February
14, 1979,
and
Concanavalin A Binding Human Fibroblasts’ ERIC
AND
(R.M.) 45267
in revised
Sites
GRUENSTEIN
University
of Cincinnati
June
20, 1979 (MS
form
College
of Medicine,
79-189)
We have Concanavalin considered
previously demonstrated the presence of a small number of high affinity binding sites for A on the plasma membrane of normal human fibroblasts. In the present study, we have the question of whether these high affinity sites are distributed uniformly on all cells or are present in high concentrations on only a small percentage of the total cell population. Using immunoelectron microscopy to observe the topological distribution of Con A on the cell surface, we have found a uniform distribution of binding sites when the cells were incubated with either 50 &g/ml Con A, a concentration sufficient to saturate both low and high affinity sites, or 0.1 &g/ml Con A, a concentration sufficient to saturate only the high affinity sites. Binding of Con A was not confined to any localized or specialized regions of the membrane. In addition, autoradiography and fluorescent microscopy techniques were used to further examine the Con A binding sites on the plasma membranes of individual cells. Again, all cells showed a uniform distribution of Con A when both high and low affinity sites were saturated. When only the high affinity sites were saturated, the absence ofany detectable binding by fluorescence or autoradiography allowed us to set quantitative lower limits on the possible levels of nonuniform binding. On the basis of these studies, combined with the immunoelectron microscopy, we have concluded that it is unlikely that there is any significant degree ofnonuniform distribution of Con A binding sites, either on the surface of a single cell or among the total population of cells. If any asymmetry exists, it must involve more than 15% of the total cell population.
Complex oligosaccharides nificant class of molecules 21).
The
study
of these
represent in biological molecules
has
an
important and cell membranes
been
facilitated
represent mined.
sig(13,
If these
by the
use of lectins, proteins which bind saccharides with high specificity (23, 7). One of these lectins is Concanavalin A (Con A), a protein isolated from the jack bean, which binds specifically to a-methyl-D-mannopyranoside or related residues of polysaccharide
chains
(11).
Con
lation ofrestinglymphocytes and cells when a small percentage of sites are occupied by the lectin which Con A produces these understood, site
to
series
but the
cell
of events
binding
A produces
mitogemc
mitogenesis, equally
ofthislectin
through
appears
stimu-
leading
to lectin-induced
its sugar
to be a necessary
step
mitogenesis
guishable techniques A binding
in the cell
plasma
agglutination (18). We have previously shown the presence of a new class of high affinity Con A binding sites on the cell surface of normal human fibroblasts (10). These sites, with a Ka = 2.2 X iO M’, close genesis
are saturated to the lectin in target
at 0.1 pig/mi concentrations cells2
(10).
Con
Whether
A, a concentration necessary to induce these
high
affinity
high
Con
affinity
A receptors
binding
they might among a population
be
remains
sites
expected of cells,
are
to be deter-
in
to be perhaps
related
to
distributed as a function
fact
unof
used three different fluorescence microsto visualize the distriCon A binding sites
among a population of randomly dividing fibroblasts (24). We reasoned that if a small number of cells contain most of the high affinity sites, then these cells should be easily distin-
binding and
mitogenic
the stage of the cell cycle. We have techniques, immunoelectron microscopy, copy and autoradiographic microscopy bution of both high and low affinity
agglutination of transformed their total number of binding (16, 19). The mechanism by biological effects is not yet
surface
the
at low
concentrations
of Con
should make it possible sites are localized on membrane
(2).
In this
work,
we
suggest that both high and low affinity are uniformly distributed on the outer membrane of all the cells.
quite mito-
MATERIALS
AND
A. In addition,
to establish specialized
whether regions
present
Con surface
data
these of
Con the
which
A binding sites of the plasma
METhODS
sites Chemicals: Lactoperoxidase and fluorescein isothiocyanate (FITC) were purchased from Sigma Chemical Co., St. Louis, Mo. Sephadex G-75 was obtained from Pharmacia Fine Chemicals Inc. Piscataway, N. J. The monosaccharide a-D-mannopyranoside was purchased from P. L. Biochemicals Inc., Milwaukee, Wisc. Horse spleen ferritin (six-times crystallized, cadmium free), goat anti-Con A and rabbit anti-goat IgG were purchased from Miles Yeda, Rehovot,
This work was supported by research grant HL-18553 from the National Institutes of Health, a Basil O’Connor Starter Research Grant from the National Foundation-March of Dimes, and a grant from the Muscular Dystrophy Association of America. 2 M Feller, WD Behnke and E Gruenstein (Biochimica Biophysics Acts, in press). I
1610
Downloaded from jhc.sagepub.com at Univ of Connecticut / Health Center / Library on June 4, 2015
CELL
Israel. Boston,
Na[’251] was obtained Mass. or Amersham
chemicals
were
obtained
from
either
Searle, from
SURFACE
DISTRIBUTION
New
Arlington
commercial
England
Heights,
OF
at
the
highest
obtainable states of purity. Concanavalin A: Con A, kindly provided by Dr. Charles Richardson of the University of Cincinnati College of Medicine, was prepared from jack bean meal (Pfaltz & Bauer, Flushing, N.Y.) by the method of Agrawal and Goldstein (1). Sodium dodecyl sulfate (SDS) polyacrylamide gels of the purified Con A revealed a single band at a mw. of 2.5 x iO daltons. Con A was stored as a lyophiized powder at 4#{176}C for up to 4 months, during which time there was no decrease in activity as determined by polysaccharide precipitation (20). [12511 Con
A, with a specific activity of 2 to 3 X iO cpm4&g protein, by the lactoperoxidase method (4). The [‘251]-labeled Con A was repurified from the reaction mixture by affinity chromatography on Sephadex G-50 equilibrated with 2.6 mM KCI, 1.46 mM was
A
overnight
Nuclear,
Ill. All other
sources,
CON
prepared
BINDING
at 4#{176}C. The
applied
to
1.5
x
30
1611
SITES
cm
following
morning
DEAE-Sephadex
the
crude
A-SO
column
globulin
was
(Pharmacia
Fine Chemicals, Upsalla, Sweden) equilibrated with 0.01 M borate buffer, pH 7.5 (8). Five milliliter fractions were collected and protein content was determined by optical density at 280 nm (Ego 14.6). Fractions possessing the greatest optical densities were pooled, dialyzed against 500 volumes of BBS, and stored at -20#{176}Cuntil needed. b) Coupling of horse spleen ferritin (HSF) to goat IgG: The conjugation of goat IgG to HSF was done by a modification of the method of dePetris and Raff (9) employing glutaraldehyde. Briefly, 5.1 mg of goat IgG and 15 mg of HSF were brought together in a total volume of 9.5 ml of PBS (0.01 M phosphate buffer with 0.14 M NaC1, pH 7.4). The solution was gently stirred for 1 hr at 4#{176}C. Conjugation was initiated by the addition of 0.5 ml of 1.0% glutaraldehyde (precooled).
After
3 hr
a PBS-lysine
(50
incubation mg
lysine
at 4#{176}C with
constant
stirring,
monohydrochloride/m.l)
were
16 ml of added
and
the solution was stirred at 4#{176}C for 8 hr. Subsequently, any large aggregates were removed by centrifugation at 1000 x g for 30 mis. In KH2PO4, 136 mM NaCI and 8 mM Na2HPO4 0.7 H20, pH 7.4 (NaClP buffer). The column was washed with 100 ml of the same buffer, order to remove any large soluble aggregates, 3.5 ml of cold saturated and the bound Con A was selectively eluted with 0.1 M a-methyl-Dammonium sulfate were added and the solution was gently stirred for mannopyranoside. The eluted material was dialyzed for 2 days at 4#{176}C 16 hr at 4#{176}C. The precipitate was removed by centrifugation at 1000 against large volumes of NaCI-P buffer. SDS polyacrylamide gel x g for 30 mis. The resultant soluble protein conjugates (supernatant electrophoresis revealed no difference between the labeled and native fluid) were concentrated by precipitation with 9.8 ml of saturated Con A preparations. The labeled lectin was stored at -20#{176}Cand used ammonium sulfate (40% final concentration). The precipitate was within 2 months. collected by centrifugation at 1500 x g for 30 min, resuspended in 2 Cells: Normal human fibroblasts (GM 43) were obtained from the ml of PBS, and dialyzed against four 500-mi changes of PBS over an Institute for Medical Research, Camden, N. J. Cells were grown in 18 hr period at 4#{176}C. Any insoluble substances were removed by monolayer and maintained in a humidified atmosphere at 37#{176}C 5% centrifugation at 1500 x g for 1 hr. Since this reaction gave products CO2-95% air. The culture medium contained Eagle’s minimum essenof three basic molecular species, i.e. goat IgG (free and polymeric), tial medium (GIBCO, no. F-li) supplemented with penicillin-strepHSF (free and polymeric), and goat IgG-HSF conjugates, further tomycin, 100 units/mI, 200 mM Tricine chloride, pH 7.4, 24 mM purification was necessary. Separation of these species was performed NaHCO:,, 1% (V/V) nonessential amino acids and 10% (V/V) fetal calf by exclusion chromatography using Sepharose 4B (3). serum. The cells were routinely checked for mycoplasma contaminac) Binding assay: All immunohistochemical reactions were done tion by standard techniques (6). They were used between the 5th and in situ in 16 x 85-mm glass Leighton tubes. Cell monolayers were 20th generations and all studies were conducted on cells in the late incubated with 0.1 or 50 &g/mi Con A as indicated. Monolayers were logarithmic phase of growth. initially fixed with 0.5% glutaraldehyde at 4#{176}C for 30 mm after which Binding of [‘25 labeled con A to cells: Cells grown to conthe cells were washed three times with NaCI-P buffer. This brief fluency in Falcon plastic tissue culture dishes (60-mm diameter) were treatment with low concentrations of glutaraldehyde has been shown removed from the incubator and placed at 4#{176}C for 30 mis. Each to crosslink proteins and leave their biological activity intact (5). The culture dish was then washed twice with cold NaC1-P buffer and cells were next incubated with goat anti-Con A (first antibody) for 30 incubated for 1 hr at 4#{176}C in 2 ml of the same buffer containing either miii at 4#{176}C. Subsequent to this incubation and three washes with cold 50 tg/mi or 0.1 zg/ml [‘251]Con A. PBS, the cells were incubated for 30 mm at 4#{176}C with rabbit anti-goat The cells were washed five times with cold NaCl-P buffer and IgO (second antibody). In this case, the first antibody becomes the dissolved in 1 ml of 0.1 N NaOH at room temperature. We have antigen for which rabbit anti-goat IgG possesses specificity. The last previously determined (data not shown) that three washes were step involves incubation of the samples with the electron dense sufficient to remove all unbound Con A. Aliquots of the 0.1 N NaOH markers, goat IgG HSF, for 30 ada at 4#{176}C. Control experiments were solubiized cells were removed and the radioactivity was measured in done in which initial exposure of cells to Con A was deleted and all a Packard
y-counter
to
determine
the
amount
of Con
A which
has
remained bound to the cells. A second aliquot was removed for protein determinations which were done according to the method of Lowry, et al. (15). Electron microscopy: a) Preparation of goat IgG: Normal goat serum was a generous gift of Dr. Beatrice Lampkin, Cincinnati Children’s Hospital, Medical Center.
Crude
parts
27% sodium
globulin
incubation
at
collected
by low
fluid
was
discarded with
0.05
To
was
added
speed
precipitated
(W/V)
23#{176}Cwith
volume this
was
sulfate
out
to one
occasional
and
the
an
equal
pellet buffer volume
by goat
agitation,
centrifugation.
M borate
part
the the
Subsequently, was with
resuspended 0.10
of 27%
addition
M NaCl, sodium
of two
After
serum.
20 mis
precipitate the to
was
supernatant the
pH sulfate
7.5
original (BBS). and
the
precipitate was collected as described. The resultant pellet was resuspended to one-half the original volume (crude globulin) in BBS and dialyzed against 500 volumes of 0.01 M borate buffer, pH 7.5 (8),
other reagents were added sequentially. In other controls 0.1 M amethyl-D-mannopyranoside was included in the incubation with Con A in order to demonstrate the sugar-specific nature of the Con A binding. After the immunohistochemical reactions, all samples were washed in
an
with ice
cold
bath.
0.1
M sodium
Postfixation
Hirsch
cacodylate was
done
buffer, according
pH
7.2,
and
to
the
method
placed of
and Fedorko (12). Briefly, this method employs the simultsneous addition of 2.5% glutaraldehyde and 1% osmium tetroxide, both buffered in 0.1 M sodium cacodylate, pH 7.2. After a 90 minute fixation, all samples were washed twice with cold cacodylate buffer, followed by two washes with physiological saline, and finally posttixed overnight with 0.5% uranyl acetate in acetate-verona! buffer at 23#{176}C. Subsequently, all samples were dehydrated, infiltrated, and embedded in Epon 812 and Araldite 6005. Polymerization of the resin was initiated by incubation overnight at 40#{176}C and completed by incubation at 60#{176}C for 72 hr. The resin containing the cell monolayer was separated from the glass by submerging the hot tube in an ice bath
Downloaded from jhc.sagepub.com at Univ of Connecticut / Health Center / Library on June 4, 2015
,
..
54
.. . ..
.,
$
I
:‘
(
-
1*A
.
‘ID
Downloaded from jhc.sagepub.com at Univ of Connecticut / Health Center / Library on June 4, 2015
,
-
CELL
(26).
Samples
cell
were
densities
cork
borer
These
(E.
blocks,
marked
microscopically, and
H. Sargent
dowels
0.5
with
cut
Company,
cm
glue
containing
out
with
were
(Borden
OF
electric
Alabama).
mounted
Company,
on
x
/4”
New
CON
York,
density clusters average colors 0.08
from
ferritin
purified
Con
A
by
buffer
pH
through
7.4
for
with
fluorescein
of Smith
and
0.15 mg of FITC
18 hr
a Sephadex
reaction method
at
G-75
4#{176}C. Free column.
(25).
in 0.1 M sodium
FITC The
isothiocyanate
Hollers
was
Con
mg
phosphate
removed
labeled
One
by
A was
passage for
agglutinating activity as previously described (20, 22). For the fluorescent studies, normal human fibroblasts were grown on glass microscope slides and incubated with either 50 or 0.1 zg/ml FITC-Cor A for 1 hr at 4#{176}C. The cells were washed four times with NaCl-P buffer to remove unbound lectin, and fixed with 3.7% formaldehyde for 20 mm at room temperature. Fluorescent microscopic examinations were carried
out
with
a
Zeiss
universal
microscope.
To
determine
the
percentage of cells showing fluorescence, at least five fields of approximately 100 cells per field were observed. Photographs were taken with Kodak high speed Ectachrome film with an exposure of 40 secs. Autoradiography: Glass slides containing monolayers of fibroblasts
were
incubated
4#{176}C. The
cells
unbound
lectin,
NaCl-P
with
were
buffer
0.1
washed
fixed and
with dried.
or
four
50 zg/ml
times
[I]
with
NaCI-P
3.7% formaldehyde, The
slides
were
Con
A for buffer
washed dipped
1 hr
at
to remove
four times
with
in a 1 :2 dilution
of
Liford L-4 liquid photographic emulsion (Polysciences Inc.) in the darkroom and allowed to air dry for 45 mis. The emulsion-coated slides were stored in light-tight boxes containing Drierite dessicant (Scientific Products, McGam Pk., Ill.) at 4#{176}C for 1-8 weeks. At the end of these time periods, the slides were developed with D-19 developer, rinsed in distilled water and fixed with Rapid-Fix (Eastman Kodak). The autoradiograms were examined with a Leitz microscope and
photographed
with
Kodak
Tri-X
film.
sites
on
distribution cell
surface:
to determine ultrastructural different
show electron A for 1 hr
at
prepared Methods.
for All
ferritin Although
grains the
labeling
was
cell
surface.
incubation sugar-specific almost
of
concanavalin
Immunoelectron
A
cells
were
micrographs 4#{176}C,washed
microscopy
immunoelectron sections of along ferritin clearly
the not
examined.
of cells with
was
exposed NaCl-P
perimeter grains were confmed
completely
cells with A binding, bare
to 50 zg/ml buffer and as large
to specialized Con the
of ferritin
lA
used
with
0. 1 pg/mi
Con
A also
the plasma of grains analysis
results
in
membrane is markedly of the grain
x
receptors =
i
cluster)
can be calculated to be 20 ferritin clusters/2.3 t 109 ferritin clusters4t2. Since the average number of grains per cluster along the membrane (5 grains! is approximately
per
we
assume
site,
thus
cluster
the
in the
that
each
giving
a
same
as
HSF-goat cluster
value
the
IgG
average
itself
represents 109
of
one
high
number
(data
affinity
not
of
shown)
Con
A binding
Con
A binding
sites4L2. From x
previous
106 high
3T3 these
mouse human
(17).
This
studies
affinity
(10)
Con
would,
we have
A sites
fibroblasts, fibroblasts,
which has
therefore,
shown
per
cell.
are been give
that
The
nor
spherical,
significantly thus bringing the
the
a theoretical
actual
greater than the calculated
observed
value.
the
density
of the
section
1.5
that the cells were cells are neither
surface
this
small
of
z2 or about a 14-fold observed value. Howby Noonan and Burger
area
is likely
to be
theoretical value of 2 x 10t2, density of Con A sites closer to
Whether
particles require and identification
3 of
per
factor
can
count for the observed discrepancy between observed densities remains unclear. Population distribution of concanavalin sites: Ferritin visualization
are area
similar in size to to be 2 x 101p2
(17) for the fibroblast surface area assumed smooth spheres. Since our surface-attached smooth,
there
surface
roughly estimated
x iO high affmity Con A receptors higher density than the experimentally ever, the theoretical value calculated
membrane
high
(
adequately
ac-
theoretical
and
A
magnification x 15,000) so
of a single
regions
grains
(Fig.
to inhibit is found 1C).
bers
of cells.
Con
A
instead of low affinity be desirable to confirm using methods capable The
binding
B
measured
in of
croscopy after tions sufficient ml) there was
and
Con then
membrane. to count,
is included A in order membrane
the 30
described numbers
of the plasma too numerous
If a-methyl-mannopyranoside of the Con
Figures
microscopy cells show
the
cells
of ferritin grains along expected, the number 1D and E). Morphometric
contain high that it would observations
binding
the distribution of Con A binding sites at level and multiple sections of more than nucleated
of the
cell
can
binding
for that be
proper only a
examined
at a time. Since there are about 170 times more low than high affinity sites, as few as 0.6% of the total cell population could
RESULTS Topological
1613
SITES
(data not shown) revealed an average of 20 ferritin per 2.3 linear length of membrane. Assuming an section thickness of 0.08 based on the interference of the sections (gold-silver), the density of Con A
grains
tested
BINDING
the appearance although, as reduced (Figs.
N.Y.). After the glue had hardened each sample was thin-sectioned with a Reichert OM-U3 Ultramicrotome using a diamond knife. All sections were picked up on uncoated 300 mesh copper grids and viewed unstained with a JEOL-iOOB (Med.ford, Mass.) electron microscope operating at 100 KV. Fluorescent Studies: Fluorescent labeled Con A was prepared (FITC) according to the of Con A was mixed with
A
Incubation
high
an
Birmingham,
in diameter,
Epoxy
DISTRIBUTION
areas
subsequently
and
measuring
wooden
3/4”
examined
were
SURFACE
by
Several
by cell
well
of the
fluorescence
in the
ized
the to be
areas
the was
of both
among
fluorescence
thousand
a series as
distribution sites
were
incubation to saturate a consistent
of
parallel
as
numerous,
(Fig.
the and
cells
high
total
examined
running
low
affinity
population
was
techniques.
by fluorescence
along
apparently
2). Whether
association
and
cell
autoradiographic
of low
these affinity
of Con
the
random
long areas
patterns binding
of membrane moving in and out microscope could not be determined a clear
felt, therefore, micrographic larger num-
ml-
with FITC-Con A. At concentrathe low affinity receptors (50 .tg/ pattern of labeling characterized
lines
concentrations
sites. We our electron of visualizing
A with
represent sites
of the here. the
axis
of the
of punctate
or are
localdue
to
plane of focus of However, there membranes
of all
FIG. 1. Electron micrographs of Concanavalin A distribution on the surface of normal human fibroblasts. Cell monolayers were exposed to Con A at 4#{176}C for 1 hr. The plates were then washed and prepared for immunoelectron microscopy as described in Materials and Methods. Panels A and B, cells exposed to 50 pg/mi Con A; panel C, cells exposed to 50 pg/mi Con A plus 10 mM a-methyl-D-mannopyranoside; panels D and E, cells exposed to 0.1 pg/mi Con A; panel E, cells exposed to 0.1 ig/ml Con A plus 0.1 mM a-methyl-D-mannopyranoside. All micrographs x 100,000.
Downloaded from jhc.sagepub.com at Univ of Connecticut / Health Center / Library on June 4, 2015
1614
FELLER,
MORRIS
AND
GRUENSTEIN
[
125i]
.4
if
.25
.5
50
00
50
200
B FIG.
the
Scatchard
3.
normal nmoles
human
of
[1251]
complex
plot
of
FITC-Concanavalin (B) and free (F) are A bound per liter.
[1251]
fibroblasts. Bound FITC-Concanavalin was
calculated
to
be
A monomer.
Scatchard
Con A yielded to the values
high and low affinity obtained in previous
In order
18 FITC
analyses
to determine
the
of the
molecules
binding
binding studies
lower
A binding expressed
per
Con
of FITC-
constants for [1251]
limits
to as
Con
[1251]
identical A (10).
of sensitivity
of the
fluorescent technique, we used decreasing concentrations of FITC-Con A in the binding assay. A progressive decrease was observed in the intensity of cell-associated fluorescence when cells were incubated with concentrations of Con A from 100 to 1 .tg/ml. Below 1 tg/mi there was no detectable fluorescence. From these data we have been able to calculate the maximum allowable degree of asymmetry of Con A distribution among the cell population. At 1 tg/ml Con A there are 2 x i0 Con A molecules bound per cell (see Fig. 3 of reference 10). This, lower limit on the number of high affinity FIG. 2. Fluorescent micrographs of FITC-Concanavalin A bound to normal human fibroblasts. Cell monolayers were incubated with 50 Lg/ml FITC-Con A for 1 hr at 4#{176}C. The cells were washed to remove unbound lectin and the amount of bound lectin was visualized with a Zeiss Photomic II microscope (A). Same cells were also visualized by
phase
contrast
microscopy
(x40
objective)
(B).
could expect that the high cell
to be able to see. affinity sites are cell. Thus, if instead of a population, high affinity
the
cells,
per
This
each
technique. cells
examined.
demonstrated Con
A in the No
these
conditions.
cence.
specificity
of
by incubation
side. washed
The
presence
of the of 10 mM
lectin-associated with
Thus,
the
with
cells
with
FITC
treated
no detectable
of FITC-Con
1 hr at 4#{176}C, no fluorescence Fluorescence photomicrographs
ity
binding
binding, Con A repurified
was
was
totally black fluorescence not
due
50 tg/mi observed
had
to
was
FITC-
alone
surface
and
fluores-
interference
exclu-
site on the FITC-Con
lectin. A for
on any of the labeled under
and are therefore under conditions of
cells. these
not shown. of high affinFITC
these
asymmetry
must
involve
are,
with
as shown in Figure 3. FITC was reacted with and the resulting FITC[I] Con A complex as described in Methods. The specific activity
the [1251]
was of
the
large
number
man
fibroblasts
would
than
able
provided
surface
of
to conclude the
the
of
were
A sites. of the
that
if there
is
affinity
sites,
it
cells.
Con
Autoradiograms
which
high
an additional
distribution
of cells.
limits
of
15%
i07 Con of sensitivity
2 x
have
the
within
distribution
more
sets a cell we
We have previously determined present at an average of 3 x 106 uniform distribution among the sites were restricted to 15% of
therefore,
of
ining
cells
above,
Autoradiography under
A is associated
detected of cells
We
any
a.methyl-D-mannopyrano-
sively with the presence of the sugar-specific When cells were incubated with 0.1 pg/mi
conditions were Failure to observe
binding
was
buffer binding
cells
A
fluorescence
Similarly,
NaCl-P
FITC-Con
of
is, as shown
therefore, sites per
incubated
method A
receptors
for
of monolayers with
exam-
among
a
of hu-
50 ,.&g/ml
[1251]
Con
A for 1 hr at 4#{176}C are shown in Figure 4A. The autoradiogram was exposed for 1 week and shows an even distribution of radioactivity surface associated by
the
inclusion
the incubation with 0.1 Lg/mi autoradiogram grains
in all cells. In a parallel experiment, radioactivity was shown to be
were
concentration. on a small
of
10
mM
a-methyl-D-mannopyranoside
medium [125!]
was observed
(Fig. 4B). Incubation Con A is shown in Fig. exposed for one month, over
If the high subpopulation
Downloaded from jhc.sagepub.com at Univ of Connecticut / Health Center / Library on June 4, 2015
the cell eliminated
any
of the
cells
affinity receptors of cells, then the
in
of cells at 4#{176}C 4C. Although the virtually no silver at
this
low
lectin
were concentrated presence of radio-
CELL
SURFACE
DISTRIBUTION
OF
CON
A
BINDING
1615
SITES
0
..
.-
‘V
‘
#{149}.:
#{176}-
0
i.
i_
.:
.
a;_ao2s;
..
#{149}
-
.
-
‘
.
.
.
.
-
-
.
.. ..‘.
“ .
-.
.
,
.
-.
_
-
_%lh
4C
..
FIG. [1251]
4.
Con
Autoradiograms A (Panel
of normal
A). with
C) for 1 hr at 4#{176}C. Autoradiograms
human
50.0 pg/mi were
[1uI]
fibroblasts Con
prepared
exposed
A in 10 mM as described
to
[I]
Concanavalin
a-methyl-D-mannopyranoside in Materials
and
A. Cell monolayers (Panel
were
B) or with
Methods.
Downloaded from jhc.sagepub.com at Univ of Connecticut / Health Center / Library on June 4, 2015
incubated
0.1 ig/ml
with [‘9]
Con
50.0 jg/ml A (Panel
1616 activity
on these
few
cells
should
have
FELLER,
MORRIS
detectable
among
been
AND affinity
the several thousand cells which we examined. The absence of such highly labeled cells is, therefore, in agreement with a uniform rather than a preferential distribution of high affinity receptors. Once
again,
minimal
level
with
these
results.
affinity Con high affinity ing
it is possible
1 out
as
that
the
A sites is about sites were located 170 cells,
intense A as do
[1251]
Con
more, (Fig.
the intensity 4A) is such
quantitatively
on Con
Given
of every
provide
to
of asymmetry
an the
ratio
affinity
6: 1000, it follows on a subpopulation then
these
that
particular
autoradiographic remaining cells
to low
if all the representcells
image at at 50 pg/mi.
time times
for the high affinity longer than that for
binding (Fig. 4A). Since autoradiographic with time (14), we should have been
of
Con
as low as Failure to our earlier
A high
binding
still we
study affinity
previously
identified
intensity is linear able to observe an
class
of high
affinity
however,
did
not
address
the
the binding of Con A occurs. affinity sites were not present
cells
high
on a small properties
only
brane
subpopulation from the
of cultured
normal
human
2%.
affinity
sites
If high
question
A
The possibility in all fibroblasts of cells having For example,
rest.
fibroblasts were
existed that but rather different memthe percentage
in mitosis
present
only
on the
is between
in these
used
affinity
to determine Con
indicate uniformly
A binding
that both distributed
the
localization
sites.
Data
on
single
showed uniform the entire cell examined affinity .1
cine
cells
are
at
the
was high
ultrastructural
distribution surface. None
then
(unpublished
R. Ph.D., observations).
University
and
here
low
This
for
conclusion
not localized important
associated
ofthe that
plasma
most
on a small to reexamine
of visualizing
example, affinity ones,
number affinity
of cells at
large
mem-
of the
high
percentage of this question numbers
if a subpopulation sites these
of cells
Since no fluorescent A and since the
sure
‘WI-Con
0.1
,Lg/ml
grains
asymmetry lation must total
over
the
A
cells,
(Fig. we
4C)
The The (10).
exists
in
or converted about 0.6%
the
cells were autoradiographic
have
of Con A binding sites stifi involve distribution
cell population. affinity Con that these sites
high
replaced represent
Thus, at Con A concentrations the high affinity receptors,
sites are saturated. 0.1 .tg/m1 FITC-Con at
of cells
have all been cells should
should label as intensely as the higher Con A concentrations
cells
failed
suffithis
small
other, where
low all
detected
at expo-
to
concluded
reveal
any
that
any
among the total cell popuamong more than 15% of
The presence of small numbers of A binding sites on the cell surface may serve a specific function. Because
of the important effects of lectins such as Con A on processes such as cell division and agglutination, it will be important to identify any specific function of Con A at the cell surface. The fact that the high affmity sites saturate at the same concentration cytes
of Con A required is provocative. In the
to induce mitogenesis present study, we have
in lymphofound that
there does not appear to be preferential association of high affinity sites to any particular region of the cell membrane. Nor does there appear to be a subpopulation of cells having a significantly higher than average number of high affinity sites our than
increase function unlikely
ability to detect such cells even ifthey 1% of the total cell population or only
represented a seven-fold
in high affinity sites per cell. We conclude that any ultimately assigned to these high affinity sites is to depend primarily on gross topological distribution. ACKNOWLEDGMENT
The authors would like to thank excellent technical assistance with
Ms. Georgianne Ciraolo the electron microscopy
1. Agrawal
technique
sites 30
LITERATURE
are
used to determine the affinity Con A binding level.
feature
for her studies.
which
binding sites on a subpopulation
of high affinity lectin of the approximately
appeared to have unusually Con A receptors. Furthermore,
Armentrout,
high
presented
high and low affinity on all cells and not
of cells. Immunoelectron microscopy topological distribution of the sites
of both
capable
techniques
despite
each mitotic cell would have approximately 3 x 10 sites. We calculated that this asymmetry should have been experimentally detectable. In this study immunoelectron, autoradiographic, and fluorescent microscopic techniques were therefore
our
using
less
1-
cells,
to be preferentially
such as fluorescence and autoradiographic microscopy. rationale used in these additional studies was as follows. ratio of high to low affinity sites is approximately 6: 1000
suggests
Con
of where
confirm were it was
very
receptors on human fibroblasts. Binding studies using radioactively labeled lectin have established the number of Con A sites present as well as their binding affinities. These techniques,
to
appear
ultrastructural
affinity receptors the cells, we felt
the
sites.
a new
brane. In order
silver
DISCUSSION We
did not
particular
of the total population. cient to saturate only
1 out of 5 cells or 20% of the total observe any autoradiographic grains conclusions of little or no asymmetry
affinity
any
which the low to high affinity
localized Further-
binding the low
receptors
with
Therefore,
image of 50 &g/ml previous studies
from
1/10 of the number of dpm/cm2 would visible as autoradiographic grains. Thus,
the exposure 4C) was four
asymmetry population. thus confirms
would
0.1 pg/mi Further-
should have been able to see high affinity receptors on about 1 out of every 20 cells in the population. more, (Fig.
the
consistent
of high
of the autoradiographic that we can estimate
(4) that less than have been clearly
estimate
A distribution
GRUENSTEIN
over cells
large Con
numbers A bound
of high to high
of Cincinnati
College
of Medi-
BL,
Goldstein
IJ:
CITED Protein-carbohydrate
interaction.
Isolation dextran 2. Anderson density
VI.
ofConcanavalin A by specific absorption on cross-linked gels. Biochim Biophys Acts 147:262, 1967 RW, Goldstein JI, Brown MS: Localization of low lipoprotein receptors on plasma membrane of normal human fibroblasts and their absence in cells from a familial hypercholesterolemia homozygote. Proc Natl Acad Sci USA 73: 2434, 1976 3. Aoki T, Boyse EA, Old U, Deharven E, HammerIng U, Wood HA: G(Gross) and H-2 cell-surface antigens: Location on gross leukemia cells by electron microscopy with visually labelled antibody.
Proc
4. Arnd-Jovin A to normal
Natl
Acad
Sci
65:569,
D, Berg P: Quantitative and transformed cells.
Downloaded from jhc.sagepub.com at Univ of Connecticut / Health Center / Library on June 4, 2015
1970
binding J Virol
of 125I Concanavalin 8:716, 1971
SURFACE
CELL
5. Avrameas
5, Ternyck
aldehyde
and
Barile
MF:
crosslinldng
of proteins
its use in the preparation 6:53, 1969
Immunochemistry
6.
T: The
Tissue
Kuise, PF and 1973, p 729
Culture
Patterson
Methods
MK,
Jr.,
DISTRIBUTION with
OF
glutar-
of immuno-absorbants. and
Applications.
Academic
Edited
Press,
New
by
York,
MM: Surface changes in transformed cells detected by lectins. Fed Proc 32:91, 1973 8. Campbell DH, Garvey JS, Cremer NE, Susedorf PH: Methods of Immunology, WA Benjamin Inc., New York, 1970, p 193 9. DePetris 5, RaffMC: Distribution ofimmunoglobin on the surface microscopy: bution and munol
2:523,
cells
as determined
by immunoferritin
Antibody induced, temperature its implications for membrane
electron
dependent structure.
Eur
redistriJ Im-
1972
10. Feller M, Richardson C, Behnke D, Gruenstein E: High and low affinity binding sites for Concanavalin A on normal human fibreblasts in vitro. Biochim. Biophys. Res. Comm. 76:1027, 1977 11. Goldstein IJ, Lucy L, Yang Y, Callies OC: Protein-carbohydrate interaction XIX. The interaction of Concanavalin A with 1gM and the glycoprotein of phytohemagglutinins of the waxbean and the soybean. J Immunol 103:695, 1969 12. Hirsch JG, Fedorko MF: Ultrastructure of human leukocytes after simultaneous fixation with glutaraldehyde and osmium tetroxide and “postflxation” in uranyl acetate. J Cell Biol 38:615, 13.
1968 Hynes
RO:
Alteration
of cell-surface
proteins
by
viral
transfor-
mation and by proteolysis. Proc Natl Acad Sci USA7O:3170, 1973 14. Laskey RA, Mills AD: Quantitative film detection of ‘H and ‘4C in polyacrylamide gels by fluorography. Eur J Biochem 56:335, 1975 15. Lowry OH, Rosebrough NJ, Farr AL Randall RJ: Protein mea-
A
BINDING
1617
SITES
surement with the folin phenol reagent. J Biol Chem 193:265, 1951 16. Nicolson GL: Neuraminidase “unmasking” and failure of trypsin to “unmask” $-D-galactose-like sites on erythrocyte, lymphoma and normal and virus-transformed fibroblasts cell membranes. J Natl
7. Burger
ofmouselymphoid
CON
Cancer
Inst
50:1443,
1973
17. Noonan normal
KD, Burger M: Binding of [3H] Concanavalin A to and transformed cells. J Biol Chem 248:4286, 1973 18. Novogrodsky A, Katchalski E: Lymphocyte transformation induced by Concanavalin A and its reversion by methyl-D-mannopryanoside. Biochim. Biophys. Acts 228:579, 1971 19. Rapin A, Burger MM: Tumor cell surfaces: general alterations detected by agglutinins. Adv Cancer Has 20:1, 1974 20. Richardson CE, Behnke WD: Physical-chemical studies on the role of metal ions in Concanavalin A. J Mol Biol 102:441, 1976 21.
Roseman
S: The
synthesis
of complex
carbohydrates
by
multi-
glycosyltransferase systems and their potential function in intracellular adhesions. Chem Phys Lipids 5:270, 1970 22. Sandvig K, Olsnes 5, Pihl A: Kinetics of Binding of the Toxic Lectins Abrin and Ricin to Surface Receptors of Human Cells. J Biol Chem 251:3977, 1976 23. Sharon N, Lis H: Lectins: Cell-agglutinating and sugar-specific proteins. Science 177:949, 1972. 24. Sharon N, Lis H: The use of lectins for the study of membranes. Methods Membr Biol 3:147, 1975. 25. Smith E, Hollers JC: The pattern of binding of fluorescein-labelled Concanavalin A to the motile lymphocyte. J Reticuloendothel Soc 8:458, 1970 26. Sutton JS: In situ embedding epoxy resin of tissue cultured in Leighton tubes; selection of single cells for electron microscopy. Stain Technol 40:151, 1965
Downloaded from jhc.sagepub.com at Univ of Connecticut / Health Center / Library on June 4, 2015
1554/79/2712-1610$02.00/0
THE
JOURNAL
Copyright
©
OF HISTOCHEMISTRY
by The
1979
Cellular
AND
Histochemical
Vol.
CYTOCHEMISTRY
Society,
Distribution of High and on the Plasma Membrane MARTHA
Departments
ofBiological
FELLER,
Chemistry
Received
for
No. 12, pp. 1610-1617, 1979 Printedin U.S.A.
27,
Inc.
(M.F.,
publication
Low Affinity of Normal
RANDAL E.G.)
MORRIS,
and Microbiology, Cincinnati, Ohio
February
14, 1979,
and
Concanavalin A Binding Human Fibroblasts’ ERIC
AND
(R.M.) 45267
in revised
Sites
GRUENSTEIN
University
of Cincinnati
June
20, 1979 (MS
form
College
of Medicine,
79-189)
We have Concanavalin considered
previously demonstrated the presence of a small number of high affinity binding sites for A on the plasma membrane of normal human fibroblasts. In the present study, we have the question of whether these high affinity sites are distributed uniformly on all cells or are present in high concentrations on only a small percentage of the total cell population. Using immunoelectron microscopy to observe the topological distribution of Con A on the cell surface, we have found a uniform distribution of binding sites when the cells were incubated with either 50 &g/ml Con A, a concentration sufficient to saturate both low and high affinity sites, or 0.1 &g/ml Con A, a concentration sufficient to saturate only the high affinity sites. Binding of Con A was not confined to any localized or specialized regions of the membrane. In addition, autoradiography and fluorescent microscopy techniques were used to further examine the Con A binding sites on the plasma membranes of individual cells. Again, all cells showed a uniform distribution of Con A when both high and low affinity sites were saturated. When only the high affinity sites were saturated, the absence ofany detectable binding by fluorescence or autoradiography allowed us to set quantitative lower limits on the possible levels of nonuniform binding. On the basis of these studies, combined with the immunoelectron microscopy, we have concluded that it is unlikely that there is any significant degree ofnonuniform distribution of Con A binding sites, either on the surface of a single cell or among the total population of cells. If any asymmetry exists, it must involve more than 15% of the total cell population.
Complex oligosaccharides nificant class of molecules 21).
The
study
of these
represent in biological molecules
has
an
important and cell membranes
been
facilitated
represent mined.
sig(13,
If these
by the
use of lectins, proteins which bind saccharides with high specificity (23, 7). One of these lectins is Concanavalin A (Con A), a protein isolated from the jack bean, which binds specifically to a-methyl-D-mannopyranoside or related residues of polysaccharide
chains
(11).
Con
lation ofrestinglymphocytes and cells when a small percentage of sites are occupied by the lectin which Con A produces these understood, site
to
series
but the
cell
of events
binding
A produces
mitogemc
mitogenesis, equally
ofthislectin
through
appears
stimu-
leading
to lectin-induced
its sugar
to be a necessary
step
mitogenesis
guishable techniques A binding
in the cell
plasma
agglutination (18). We have previously shown the presence of a new class of high affinity Con A binding sites on the cell surface of normal human fibroblasts (10). These sites, with a Ka = 2.2 X iO M’, close genesis
are saturated to the lectin in target
at 0.1 pig/mi concentrations cells2
(10).
Con
Whether
A, a concentration necessary to induce these
high
affinity
high
Con
affinity
A receptors
binding
they might among a population
be
remains
sites
expected of cells,
are
to be deter-
in
to be perhaps
related
to
distributed as a function
fact
unof
used three different fluorescence microsto visualize the distriCon A binding sites
among a population of randomly dividing fibroblasts (24). We reasoned that if a small number of cells contain most of the high affinity sites, then these cells should be easily distin-
binding and
mitogenic
the stage of the cell cycle. We have techniques, immunoelectron microscopy, copy and autoradiographic microscopy bution of both high and low affinity
agglutination of transformed their total number of binding (16, 19). The mechanism by biological effects is not yet
surface
the
at low
concentrations
of Con
should make it possible sites are localized on membrane
(2).
In this
work,
we
suggest that both high and low affinity are uniformly distributed on the outer membrane of all the cells.
quite mito-
MATERIALS
AND
A. In addition,
to establish specialized
whether regions
present
Con surface
data
these of
Con the
which
A binding sites of the plasma
METhODS
sites Chemicals: Lactoperoxidase and fluorescein isothiocyanate (FITC) were purchased from Sigma Chemical Co., St. Louis, Mo. Sephadex G-75 was obtained from Pharmacia Fine Chemicals Inc. Piscataway, N. J. The monosaccharide a-D-mannopyranoside was purchased from P. L. Biochemicals Inc., Milwaukee, Wisc. Horse spleen ferritin (six-times crystallized, cadmium free), goat anti-Con A and rabbit anti-goat IgG were purchased from Miles Yeda, Rehovot,
This work was supported by research grant HL-18553 from the National Institutes of Health, a Basil O’Connor Starter Research Grant from the National Foundation-March of Dimes, and a grant from the Muscular Dystrophy Association of America. 2 M Feller, WD Behnke and E Gruenstein (Biochimica Biophysics Acts, in press). I
1610
Downloaded from jhc.sagepub.com at Univ of Connecticut / Health Center / Library on June 4, 2015
CELL
Israel. Boston,
Na[’251] was obtained Mass. or Amersham
chemicals
were
obtained
from
either
Searle, from
SURFACE
DISTRIBUTION
New
Arlington
commercial
England
Heights,
OF
at
the
highest
obtainable states of purity. Concanavalin A: Con A, kindly provided by Dr. Charles Richardson of the University of Cincinnati College of Medicine, was prepared from jack bean meal (Pfaltz & Bauer, Flushing, N.Y.) by the method of Agrawal and Goldstein (1). Sodium dodecyl sulfate (SDS) polyacrylamide gels of the purified Con A revealed a single band at a mw. of 2.5 x iO daltons. Con A was stored as a lyophiized powder at 4#{176}C for up to 4 months, during which time there was no decrease in activity as determined by polysaccharide precipitation (20). [12511 Con
A, with a specific activity of 2 to 3 X iO cpm4&g protein, by the lactoperoxidase method (4). The [‘251]-labeled Con A was repurified from the reaction mixture by affinity chromatography on Sephadex G-50 equilibrated with 2.6 mM KCI, 1.46 mM was
A
overnight
Nuclear,
Ill. All other
sources,
CON
prepared
BINDING
at 4#{176}C. The
applied
to
1.5
x
30
1611
SITES
cm
following
morning
DEAE-Sephadex
the
crude
A-SO
column
globulin
was
(Pharmacia
Fine Chemicals, Upsalla, Sweden) equilibrated with 0.01 M borate buffer, pH 7.5 (8). Five milliliter fractions were collected and protein content was determined by optical density at 280 nm (Ego 14.6). Fractions possessing the greatest optical densities were pooled, dialyzed against 500 volumes of BBS, and stored at -20#{176}Cuntil needed. b) Coupling of horse spleen ferritin (HSF) to goat IgG: The conjugation of goat IgG to HSF was done by a modification of the method of dePetris and Raff (9) employing glutaraldehyde. Briefly, 5.1 mg of goat IgG and 15 mg of HSF were brought together in a total volume of 9.5 ml of PBS (0.01 M phosphate buffer with 0.14 M NaC1, pH 7.4). The solution was gently stirred for 1 hr at 4#{176}C. Conjugation was initiated by the addition of 0.5 ml of 1.0% glutaraldehyde (precooled).
After
3 hr
a PBS-lysine
(50
incubation mg
lysine
at 4#{176}C with
constant
stirring,
monohydrochloride/m.l)
were
16 ml of added
and
the solution was stirred at 4#{176}C for 8 hr. Subsequently, any large aggregates were removed by centrifugation at 1000 x g for 30 mis. In KH2PO4, 136 mM NaCI and 8 mM Na2HPO4 0.7 H20, pH 7.4 (NaClP buffer). The column was washed with 100 ml of the same buffer, order to remove any large soluble aggregates, 3.5 ml of cold saturated and the bound Con A was selectively eluted with 0.1 M a-methyl-Dammonium sulfate were added and the solution was gently stirred for mannopyranoside. The eluted material was dialyzed for 2 days at 4#{176}C 16 hr at 4#{176}C. The precipitate was removed by centrifugation at 1000 against large volumes of NaCI-P buffer. SDS polyacrylamide gel x g for 30 mis. The resultant soluble protein conjugates (supernatant electrophoresis revealed no difference between the labeled and native fluid) were concentrated by precipitation with 9.8 ml of saturated Con A preparations. The labeled lectin was stored at -20#{176}Cand used ammonium sulfate (40% final concentration). The precipitate was within 2 months. collected by centrifugation at 1500 x g for 30 min, resuspended in 2 Cells: Normal human fibroblasts (GM 43) were obtained from the ml of PBS, and dialyzed against four 500-mi changes of PBS over an Institute for Medical Research, Camden, N. J. Cells were grown in 18 hr period at 4#{176}C. Any insoluble substances were removed by monolayer and maintained in a humidified atmosphere at 37#{176}C 5% centrifugation at 1500 x g for 1 hr. Since this reaction gave products CO2-95% air. The culture medium contained Eagle’s minimum essenof three basic molecular species, i.e. goat IgG (free and polymeric), tial medium (GIBCO, no. F-li) supplemented with penicillin-strepHSF (free and polymeric), and goat IgG-HSF conjugates, further tomycin, 100 units/mI, 200 mM Tricine chloride, pH 7.4, 24 mM purification was necessary. Separation of these species was performed NaHCO:,, 1% (V/V) nonessential amino acids and 10% (V/V) fetal calf by exclusion chromatography using Sepharose 4B (3). serum. The cells were routinely checked for mycoplasma contaminac) Binding assay: All immunohistochemical reactions were done tion by standard techniques (6). They were used between the 5th and in situ in 16 x 85-mm glass Leighton tubes. Cell monolayers were 20th generations and all studies were conducted on cells in the late incubated with 0.1 or 50 &g/mi Con A as indicated. Monolayers were logarithmic phase of growth. initially fixed with 0.5% glutaraldehyde at 4#{176}C for 30 mm after which Binding of [‘25 labeled con A to cells: Cells grown to conthe cells were washed three times with NaCI-P buffer. This brief fluency in Falcon plastic tissue culture dishes (60-mm diameter) were treatment with low concentrations of glutaraldehyde has been shown removed from the incubator and placed at 4#{176}C for 30 mis. Each to crosslink proteins and leave their biological activity intact (5). The culture dish was then washed twice with cold NaC1-P buffer and cells were next incubated with goat anti-Con A (first antibody) for 30 incubated for 1 hr at 4#{176}C in 2 ml of the same buffer containing either miii at 4#{176}C. Subsequent to this incubation and three washes with cold 50 tg/mi or 0.1 zg/ml [‘251]Con A. PBS, the cells were incubated for 30 mm at 4#{176}C with rabbit anti-goat The cells were washed five times with cold NaCl-P buffer and IgO (second antibody). In this case, the first antibody becomes the dissolved in 1 ml of 0.1 N NaOH at room temperature. We have antigen for which rabbit anti-goat IgG possesses specificity. The last previously determined (data not shown) that three washes were step involves incubation of the samples with the electron dense sufficient to remove all unbound Con A. Aliquots of the 0.1 N NaOH markers, goat IgG HSF, for 30 ada at 4#{176}C. Control experiments were solubiized cells were removed and the radioactivity was measured in done in which initial exposure of cells to Con A was deleted and all a Packard
y-counter
to
determine
the
amount
of Con
A which
has
remained bound to the cells. A second aliquot was removed for protein determinations which were done according to the method of Lowry, et al. (15). Electron microscopy: a) Preparation of goat IgG: Normal goat serum was a generous gift of Dr. Beatrice Lampkin, Cincinnati Children’s Hospital, Medical Center.
Crude
parts
27% sodium
globulin
incubation
at
collected
by low
fluid
was
discarded with
0.05
To
was
added
speed
precipitated
(W/V)
23#{176}Cwith
volume this
was
sulfate
out
to one
occasional
and
the
an
equal
pellet buffer volume
by goat
agitation,
centrifugation.
M borate
part
the the
Subsequently, was with
resuspended 0.10
of 27%
addition
M NaCl, sodium
of two
After
serum.
20 mis
precipitate the to
was
supernatant the
pH sulfate
7.5
original (BBS). and
the
precipitate was collected as described. The resultant pellet was resuspended to one-half the original volume (crude globulin) in BBS and dialyzed against 500 volumes of 0.01 M borate buffer, pH 7.5 (8),
other reagents were added sequentially. In other controls 0.1 M amethyl-D-mannopyranoside was included in the incubation with Con A in order to demonstrate the sugar-specific nature of the Con A binding. After the immunohistochemical reactions, all samples were washed in
an
with ice
cold
bath.
0.1
M sodium
Postfixation
Hirsch
cacodylate was
done
buffer, according
pH
7.2,
and
to
the
method
placed of
and Fedorko (12). Briefly, this method employs the simultsneous addition of 2.5% glutaraldehyde and 1% osmium tetroxide, both buffered in 0.1 M sodium cacodylate, pH 7.2. After a 90 minute fixation, all samples were washed twice with cold cacodylate buffer, followed by two washes with physiological saline, and finally posttixed overnight with 0.5% uranyl acetate in acetate-verona! buffer at 23#{176}C. Subsequently, all samples were dehydrated, infiltrated, and embedded in Epon 812 and Araldite 6005. Polymerization of the resin was initiated by incubation overnight at 40#{176}C and completed by incubation at 60#{176}C for 72 hr. The resin containing the cell monolayer was separated from the glass by submerging the hot tube in an ice bath
Downloaded from jhc.sagepub.com at Univ of Connecticut / Health Center / Library on June 4, 2015
,
..
54
.. . ..
.,
$
I
:‘
(
-
1*A
.
‘ID
Downloaded from jhc.sagepub.com at Univ of Connecticut / Health Center / Library on June 4, 2015
,
-
CELL
(26).
Samples
cell
were
densities
cork
borer
These
(E.
blocks,
marked
microscopically, and
H. Sargent
dowels
0.5
with
cut
Company,
cm
glue
containing
out
with
were
(Borden
OF
electric
Alabama).
mounted
Company,
on
x
/4”
New
CON
York,
density clusters average colors 0.08
from
ferritin
purified
Con
A
by
buffer
pH
through
7.4
for
with
fluorescein
of Smith
and
0.15 mg of FITC
18 hr
a Sephadex
reaction method
at
G-75
4#{176}C. Free column.
(25).
in 0.1 M sodium
FITC The
isothiocyanate
Hollers
was
Con
mg
phosphate
removed
labeled
One
by
A was
passage for
agglutinating activity as previously described (20, 22). For the fluorescent studies, normal human fibroblasts were grown on glass microscope slides and incubated with either 50 or 0.1 zg/ml FITC-Cor A for 1 hr at 4#{176}C. The cells were washed four times with NaCl-P buffer to remove unbound lectin, and fixed with 3.7% formaldehyde for 20 mm at room temperature. Fluorescent microscopic examinations were carried
out
with
a
Zeiss
universal
microscope.
To
determine
the
percentage of cells showing fluorescence, at least five fields of approximately 100 cells per field were observed. Photographs were taken with Kodak high speed Ectachrome film with an exposure of 40 secs. Autoradiography: Glass slides containing monolayers of fibroblasts
were
incubated
4#{176}C. The
cells
unbound
lectin,
NaCl-P
with
were
buffer
0.1
washed
fixed and
with dried.
or
four
50 zg/ml
times
[I]
with
NaCI-P
3.7% formaldehyde, The
slides
were
Con
A for buffer
washed dipped
1 hr
at
to remove
four times
with
in a 1 :2 dilution
of
Liford L-4 liquid photographic emulsion (Polysciences Inc.) in the darkroom and allowed to air dry for 45 mis. The emulsion-coated slides were stored in light-tight boxes containing Drierite dessicant (Scientific Products, McGam Pk., Ill.) at 4#{176}C for 1-8 weeks. At the end of these time periods, the slides were developed with D-19 developer, rinsed in distilled water and fixed with Rapid-Fix (Eastman Kodak). The autoradiograms were examined with a Leitz microscope and
photographed
with
Kodak
Tri-X
film.
sites
on
distribution cell
surface:
to determine ultrastructural different
show electron A for 1 hr
at
prepared Methods.
for All
ferritin Although
grains the
labeling
was
cell
surface.
incubation sugar-specific almost
of
concanavalin
Immunoelectron
A
cells
were
micrographs 4#{176}C,washed
microscopy
immunoelectron sections of along ferritin clearly
the not
examined.
of cells with
was
exposed NaCl-P
perimeter grains were confmed
completely
cells with A binding, bare
to 50 zg/ml buffer and as large
to specialized Con the
of ferritin
lA
used
with
0. 1 pg/mi
Con
A also
the plasma of grains analysis
results
in
membrane is markedly of the grain
x
receptors =
i
cluster)
can be calculated to be 20 ferritin clusters/2.3 t 109 ferritin clusters4t2. Since the average number of grains per cluster along the membrane (5 grains! is approximately
per
we
assume
site,
thus
cluster
the
in the
that
each
giving
a
same
as
HSF-goat cluster
value
the
IgG
average
itself
represents 109
of
one
high
number
(data
affinity
not
of
shown)
Con
A binding
Con
A binding
sites4L2. From x
previous
106 high
3T3 these
mouse human
(17).
This
studies
affinity
(10)
Con
would,
we have
A sites
fibroblasts, fibroblasts,
which has
therefore,
shown
per
cell.
are been give
that
The
nor
spherical,
significantly thus bringing the
the
a theoretical
actual
greater than the calculated
observed
value.
the
density
of the
section
1.5
that the cells were cells are neither
surface
this
small
of
z2 or about a 14-fold observed value. Howby Noonan and Burger
area
is likely
to be
theoretical value of 2 x 10t2, density of Con A sites closer to
Whether
particles require and identification
3 of
per
factor
can
count for the observed discrepancy between observed densities remains unclear. Population distribution of concanavalin sites: Ferritin visualization
are area
similar in size to to be 2 x 101p2
(17) for the fibroblast surface area assumed smooth spheres. Since our surface-attached smooth,
there
surface
roughly estimated
x iO high affmity Con A receptors higher density than the experimentally ever, the theoretical value calculated
membrane
high
(
adequately
ac-
theoretical
and
A
magnification x 15,000) so
of a single
regions
grains
(Fig.
to inhibit is found 1C).
bers
of cells.
Con
A
instead of low affinity be desirable to confirm using methods capable The
binding
B
measured
in of
croscopy after tions sufficient ml) there was
and
Con then
membrane. to count,
is included A in order membrane
the 30
described numbers
of the plasma too numerous
If a-methyl-mannopyranoside of the Con
Figures
microscopy cells show
the
cells
of ferritin grains along expected, the number 1D and E). Morphometric
contain high that it would observations
binding
the distribution of Con A binding sites at level and multiple sections of more than nucleated
of the
cell
can
binding
for that be
proper only a
examined
at a time. Since there are about 170 times more low than high affinity sites, as few as 0.6% of the total cell population could
RESULTS Topological
1613
SITES
(data not shown) revealed an average of 20 ferritin per 2.3 linear length of membrane. Assuming an section thickness of 0.08 based on the interference of the sections (gold-silver), the density of Con A
grains
tested
BINDING
the appearance although, as reduced (Figs.
N.Y.). After the glue had hardened each sample was thin-sectioned with a Reichert OM-U3 Ultramicrotome using a diamond knife. All sections were picked up on uncoated 300 mesh copper grids and viewed unstained with a JEOL-iOOB (Med.ford, Mass.) electron microscope operating at 100 KV. Fluorescent Studies: Fluorescent labeled Con A was prepared (FITC) according to the of Con A was mixed with
A
Incubation
high
an
Birmingham,
in diameter,
Epoxy
DISTRIBUTION
areas
subsequently
and
measuring
wooden
3/4”
examined
were
SURFACE
by
Several
by cell
well
of the
fluorescence
in the
ized
the to be
areas
the was
of both
among
fluorescence
thousand
a series as
distribution sites
were
incubation to saturate a consistent
of
parallel
as
numerous,
(Fig.
the and
cells
high
total
examined
running
low
affinity
population
was
techniques.
by fluorescence
along
apparently
2). Whether
association
and
cell
autoradiographic
of low
these affinity
of Con
the
random
long areas
patterns binding
of membrane moving in and out microscope could not be determined a clear
felt, therefore, micrographic larger num-
ml-
with FITC-Con A. At concentrathe low affinity receptors (50 .tg/ pattern of labeling characterized
lines
concentrations
sites. We our electron of visualizing
A with
represent sites
of the here. the
axis
of the
of punctate
or are
localdue
to
plane of focus of However, there membranes
of all
FIG. 1. Electron micrographs of Concanavalin A distribution on the surface of normal human fibroblasts. Cell monolayers were exposed to Con A at 4#{176}C for 1 hr. The plates were then washed and prepared for immunoelectron microscopy as described in Materials and Methods. Panels A and B, cells exposed to 50 pg/mi Con A; panel C, cells exposed to 50 pg/mi Con A plus 10 mM a-methyl-D-mannopyranoside; panels D and E, cells exposed to 0.1 pg/mi Con A; panel E, cells exposed to 0.1 ig/ml Con A plus 0.1 mM a-methyl-D-mannopyranoside. All micrographs x 100,000.
Downloaded from jhc.sagepub.com at Univ of Connecticut / Health Center / Library on June 4, 2015
1614
FELLER,
MORRIS
AND
GRUENSTEIN
[
125i]
.4
if
.25
.5
50
00
50
200
B FIG.
the
Scatchard
3.
normal nmoles
human
of
[1251]
complex
plot
of
FITC-Concanavalin (B) and free (F) are A bound per liter.
[1251]
fibroblasts. Bound FITC-Concanavalin was
calculated
to
be
A monomer.
Scatchard
Con A yielded to the values
high and low affinity obtained in previous
In order
18 FITC
analyses
to determine
the
of the
molecules
binding
binding studies
lower
A binding expressed
per
Con
of FITC-
constants for [1251]
limits
to as
Con
[1251]
identical A (10).
of sensitivity
of the
fluorescent technique, we used decreasing concentrations of FITC-Con A in the binding assay. A progressive decrease was observed in the intensity of cell-associated fluorescence when cells were incubated with concentrations of Con A from 100 to 1 .tg/ml. Below 1 tg/mi there was no detectable fluorescence. From these data we have been able to calculate the maximum allowable degree of asymmetry of Con A distribution among the cell population. At 1 tg/ml Con A there are 2 x i0 Con A molecules bound per cell (see Fig. 3 of reference 10). This, lower limit on the number of high affinity FIG. 2. Fluorescent micrographs of FITC-Concanavalin A bound to normal human fibroblasts. Cell monolayers were incubated with 50 Lg/ml FITC-Con A for 1 hr at 4#{176}C. The cells were washed to remove unbound lectin and the amount of bound lectin was visualized with a Zeiss Photomic II microscope (A). Same cells were also visualized by
phase
contrast
microscopy
(x40
objective)
(B).
could expect that the high cell
to be able to see. affinity sites are cell. Thus, if instead of a population, high affinity
the
cells,
per
This
each
technique. cells
examined.
demonstrated Con
A in the No
these
conditions.
cence.
specificity
of
by incubation
side. washed
The
presence
of the of 10 mM
lectin-associated with
Thus,
the
with
cells
with
FITC
treated
no detectable
of FITC-Con
1 hr at 4#{176}C, no fluorescence Fluorescence photomicrographs
ity
binding
binding, Con A repurified
was
was
totally black fluorescence not
due
50 tg/mi observed
had
to
was
FITC-
alone
surface
and
fluores-
interference
exclu-
site on the FITC-Con
lectin. A for
on any of the labeled under
and are therefore under conditions of
cells. these
not shown. of high affinFITC
these
asymmetry
must
involve
are,
with
as shown in Figure 3. FITC was reacted with and the resulting FITC[I] Con A complex as described in Methods. The specific activity
the [1251]
was of
the
large
number
man
fibroblasts
would
than
able
provided
surface
of
to conclude the
the
of
were
A sites. of the
that
if there
is
affinity
sites,
it
cells.
Con
Autoradiograms
which
high
an additional
distribution
of cells.
limits
of
15%
i07 Con of sensitivity
2 x
have
the
within
distribution
more
sets a cell we
We have previously determined present at an average of 3 x 106 uniform distribution among the sites were restricted to 15% of
therefore,
of
ining
cells
above,
Autoradiography under
A is associated
detected of cells
We
any
a.methyl-D-mannopyrano-
sively with the presence of the sugar-specific When cells were incubated with 0.1 pg/mi
conditions were Failure to observe
binding
was
buffer binding
cells
A
fluorescence
Similarly,
NaCl-P
FITC-Con
of
is, as shown
therefore, sites per
incubated
method A
receptors
for
of monolayers with
exam-
among
a
of hu-
50 ,.&g/ml
[1251]
Con
A for 1 hr at 4#{176}C are shown in Figure 4A. The autoradiogram was exposed for 1 week and shows an even distribution of radioactivity surface associated by
the
inclusion
the incubation with 0.1 Lg/mi autoradiogram grains
in all cells. In a parallel experiment, radioactivity was shown to be
were
concentration. on a small
of
10
mM
a-methyl-D-mannopyranoside
medium [125!]
was observed
(Fig. 4B). Incubation Con A is shown in Fig. exposed for one month, over
If the high subpopulation
Downloaded from jhc.sagepub.com at Univ of Connecticut / Health Center / Library on June 4, 2015
the cell eliminated
any
of the
cells
affinity receptors of cells, then the
in
of cells at 4#{176}C 4C. Although the virtually no silver at
this
low
lectin
were concentrated presence of radio-
CELL
SURFACE
DISTRIBUTION
OF
CON
A
BINDING
1615
SITES
0
..
.-
‘V
‘
#{149}.:
#{176}-
0
i.
i_
.:
.
a;_ao2s;
..
#{149}
-
.
-
‘
.
.
.
.
-
-
.
.. ..‘.
“ .
-.
.
,
.
-.
_
-
_%lh
4C
..
FIG. [1251]
4.
Con
Autoradiograms A (Panel
of normal
A). with
C) for 1 hr at 4#{176}C. Autoradiograms
human
50.0 pg/mi were
[1uI]
fibroblasts Con
prepared
exposed
A in 10 mM as described
to
[I]
Concanavalin
a-methyl-D-mannopyranoside in Materials
and
A. Cell monolayers (Panel
were
B) or with
Methods.
Downloaded from jhc.sagepub.com at Univ of Connecticut / Health Center / Library on June 4, 2015
incubated
0.1 ig/ml
with [‘9]
Con
50.0 jg/ml A (Panel
1616 activity
on these
few
cells
should
have
FELLER,
MORRIS
detectable
among
been
AND affinity
the several thousand cells which we examined. The absence of such highly labeled cells is, therefore, in agreement with a uniform rather than a preferential distribution of high affinity receptors. Once
again,
minimal
level
with
these
results.
affinity Con high affinity ing
it is possible
1 out
as
that
the
A sites is about sites were located 170 cells,
intense A as do
[1251]
Con
more, (Fig.
the intensity 4A) is such
quantitatively
on Con
Given
of every
provide
to
of asymmetry
an the
ratio
affinity
6: 1000, it follows on a subpopulation then
these
that
particular
autoradiographic remaining cells
to low
if all the representcells
image at at 50 pg/mi.
time times
for the high affinity longer than that for
binding (Fig. 4A). Since autoradiographic with time (14), we should have been
of
Con
as low as Failure to our earlier
A high
binding
still we
study affinity
previously
identified
intensity is linear able to observe an
class
of high
affinity
however,
did
not
address
the
the binding of Con A occurs. affinity sites were not present
cells
high
on a small properties
only
brane
subpopulation from the
of cultured
normal
human
2%.
affinity
sites
If high
question
A
The possibility in all fibroblasts of cells having For example,
rest.
fibroblasts were
existed that but rather different memthe percentage
in mitosis
present
only
on the
is between
in these
used
affinity
to determine Con
indicate uniformly
A binding
that both distributed
the
localization
sites.
Data
on
single
showed uniform the entire cell examined affinity .1
cine
cells
are
at
the
was high
ultrastructural
distribution surface. None
then
(unpublished
R. Ph.D., observations).
University
and
here
low
This
for
conclusion
not localized important
associated
ofthe that
plasma
most
on a small to reexamine
of visualizing
example, affinity ones,
number affinity
of cells at
large
mem-
of the
high
percentage of this question numbers
if a subpopulation sites these
of cells
Since no fluorescent A and since the
sure
‘WI-Con
0.1
,Lg/ml
grains
asymmetry lation must total
over
the
A
cells,
(Fig. we
4C)
The The (10).
exists
in
or converted about 0.6%
the
cells were autoradiographic
have
of Con A binding sites stifi involve distribution
cell population. affinity Con that these sites
high
replaced represent
Thus, at Con A concentrations the high affinity receptors,
sites are saturated. 0.1 .tg/m1 FITC-Con at
of cells
have all been cells should
should label as intensely as the higher Con A concentrations
cells
failed
suffithis
small
other, where
low all
detected
at expo-
to
concluded
reveal
any
that
any
among the total cell popuamong more than 15% of
The presence of small numbers of A binding sites on the cell surface may serve a specific function. Because
of the important effects of lectins such as Con A on processes such as cell division and agglutination, it will be important to identify any specific function of Con A at the cell surface. The fact that the high affmity sites saturate at the same concentration cytes
of Con A required is provocative. In the
to induce mitogenesis present study, we have
in lymphofound that
there does not appear to be preferential association of high affinity sites to any particular region of the cell membrane. Nor does there appear to be a subpopulation of cells having a significantly higher than average number of high affinity sites our than
increase function unlikely
ability to detect such cells even ifthey 1% of the total cell population or only
represented a seven-fold
in high affinity sites per cell. We conclude that any ultimately assigned to these high affinity sites is to depend primarily on gross topological distribution. ACKNOWLEDGMENT
The authors would like to thank excellent technical assistance with
Ms. Georgianne Ciraolo the electron microscopy
1. Agrawal
technique
sites 30
LITERATURE
are
used to determine the affinity Con A binding level.
feature
for her studies.
which
binding sites on a subpopulation
of high affinity lectin of the approximately
appeared to have unusually Con A receptors. Furthermore,
Armentrout,
high
presented
high and low affinity on all cells and not
of cells. Immunoelectron microscopy topological distribution of the sites
of both
capable
techniques
despite
each mitotic cell would have approximately 3 x 10 sites. We calculated that this asymmetry should have been experimentally detectable. In this study immunoelectron, autoradiographic, and fluorescent microscopic techniques were therefore
our
using
less
1-
cells,
to be preferentially
such as fluorescence and autoradiographic microscopy. rationale used in these additional studies was as follows. ratio of high to low affinity sites is approximately 6: 1000
suggests
Con
of where
confirm were it was
very
receptors on human fibroblasts. Binding studies using radioactively labeled lectin have established the number of Con A sites present as well as their binding affinities. These techniques,
to
appear
ultrastructural
affinity receptors the cells, we felt
the
sites.
a new
brane. In order
silver
DISCUSSION We
did not
particular
of the total population. cient to saturate only
1 out of 5 cells or 20% of the total observe any autoradiographic grains conclusions of little or no asymmetry
affinity
any
which the low to high affinity
localized Further-
binding the low
receptors
with
Therefore,
image of 50 &g/ml previous studies
from
1/10 of the number of dpm/cm2 would visible as autoradiographic grains. Thus,
the exposure 4C) was four
asymmetry population. thus confirms
would
0.1 pg/mi Further-
should have been able to see high affinity receptors on about 1 out of every 20 cells in the population. more, (Fig.
the
consistent
of high
of the autoradiographic that we can estimate
(4) that less than have been clearly
estimate
A distribution
GRUENSTEIN
over cells
large Con
numbers A bound
of high to high
of Cincinnati
College
of Medi-
BL,
Goldstein
IJ:
CITED Protein-carbohydrate
interaction.
Isolation dextran 2. Anderson density
VI.
ofConcanavalin A by specific absorption on cross-linked gels. Biochim Biophys Acts 147:262, 1967 RW, Goldstein JI, Brown MS: Localization of low lipoprotein receptors on plasma membrane of normal human fibroblasts and their absence in cells from a familial hypercholesterolemia homozygote. Proc Natl Acad Sci USA 73: 2434, 1976 3. Aoki T, Boyse EA, Old U, Deharven E, HammerIng U, Wood HA: G(Gross) and H-2 cell-surface antigens: Location on gross leukemia cells by electron microscopy with visually labelled antibody.
Proc
4. Arnd-Jovin A to normal
Natl
Acad
Sci
65:569,
D, Berg P: Quantitative and transformed cells.
Downloaded from jhc.sagepub.com at Univ of Connecticut / Health Center / Library on June 4, 2015
1970
binding J Virol
of 125I Concanavalin 8:716, 1971
SURFACE
CELL
5. Avrameas
5, Ternyck
aldehyde
and
Barile
MF:
crosslinldng
of proteins
its use in the preparation 6:53, 1969
Immunochemistry
6.
T: The
Tissue
Kuise, PF and 1973, p 729
Culture
Patterson
Methods
MK,
Jr.,
DISTRIBUTION with
OF
glutar-
of immuno-absorbants. and
Applications.
Academic
Edited
Press,
New
by
York,
MM: Surface changes in transformed cells detected by lectins. Fed Proc 32:91, 1973 8. Campbell DH, Garvey JS, Cremer NE, Susedorf PH: Methods of Immunology, WA Benjamin Inc., New York, 1970, p 193 9. DePetris 5, RaffMC: Distribution ofimmunoglobin on the surface microscopy: bution and munol
2:523,
cells
as determined
by immunoferritin
Antibody induced, temperature its implications for membrane
electron
dependent structure.
Eur
redistriJ Im-
1972
10. Feller M, Richardson C, Behnke D, Gruenstein E: High and low affinity binding sites for Concanavalin A on normal human fibreblasts in vitro. Biochim. Biophys. Res. Comm. 76:1027, 1977 11. Goldstein IJ, Lucy L, Yang Y, Callies OC: Protein-carbohydrate interaction XIX. The interaction of Concanavalin A with 1gM and the glycoprotein of phytohemagglutinins of the waxbean and the soybean. J Immunol 103:695, 1969 12. Hirsch JG, Fedorko MF: Ultrastructure of human leukocytes after simultaneous fixation with glutaraldehyde and osmium tetroxide and “postflxation” in uranyl acetate. J Cell Biol 38:615, 13.
1968 Hynes
RO:
Alteration
of cell-surface
proteins
by
viral
transfor-
mation and by proteolysis. Proc Natl Acad Sci USA7O:3170, 1973 14. Laskey RA, Mills AD: Quantitative film detection of ‘H and ‘4C in polyacrylamide gels by fluorography. Eur J Biochem 56:335, 1975 15. Lowry OH, Rosebrough NJ, Farr AL Randall RJ: Protein mea-
A
BINDING
1617
SITES
surement with the folin phenol reagent. J Biol Chem 193:265, 1951 16. Nicolson GL: Neuraminidase “unmasking” and failure of trypsin to “unmask” $-D-galactose-like sites on erythrocyte, lymphoma and normal and virus-transformed fibroblasts cell membranes. J Natl
7. Burger
ofmouselymphoid
CON
Cancer
Inst
50:1443,
1973
17. Noonan normal
KD, Burger M: Binding of [3H] Concanavalin A to and transformed cells. J Biol Chem 248:4286, 1973 18. Novogrodsky A, Katchalski E: Lymphocyte transformation induced by Concanavalin A and its reversion by methyl-D-mannopryanoside. Biochim. Biophys. Acts 228:579, 1971 19. Rapin A, Burger MM: Tumor cell surfaces: general alterations detected by agglutinins. Adv Cancer Has 20:1, 1974 20. Richardson CE, Behnke WD: Physical-chemical studies on the role of metal ions in Concanavalin A. J Mol Biol 102:441, 1976 21.
Roseman
S: The
synthesis
of complex
carbohydrates
by
multi-
glycosyltransferase systems and their potential function in intracellular adhesions. Chem Phys Lipids 5:270, 1970 22. Sandvig K, Olsnes 5, Pihl A: Kinetics of Binding of the Toxic Lectins Abrin and Ricin to Surface Receptors of Human Cells. J Biol Chem 251:3977, 1976 23. Sharon N, Lis H: Lectins: Cell-agglutinating and sugar-specific proteins. Science 177:949, 1972. 24. Sharon N, Lis H: The use of lectins for the study of membranes. Methods Membr Biol 3:147, 1975. 25. Smith E, Hollers JC: The pattern of binding of fluorescein-labelled Concanavalin A to the motile lymphocyte. J Reticuloendothel Soc 8:458, 1970 26. Sutton JS: In situ embedding epoxy resin of tissue cultured in Leighton tubes; selection of single cells for electron microscopy. Stain Technol 40:151, 1965
Downloaded from jhc.sagepub.com at Univ of Connecticut / Health Center / Library on June 4, 2015
