CALIFORNIA STATE UNIVERSITY, NORTHRIDGE
THE DISPLAY OF LECTIN RECEPTOR SITES ON THE SURFACES
\\
OF TUMOR AND Et1BRYONIC CELL POPULATIONS
A thesis submitted in partial satisfaction of the
requirements for the degree of ~laster of Science in
Biology
by
Anthony Neri
~
August, 1976
California State University, Northridge
;i
ACKNOVJLEOGMENT
I would like to thank Drs. Donald Bianchi and Kenneth Jones
for the help they have provided, to Marie Roberson and Dan Connolly
for their assistance, to.Dr. Steven B. Oppenheimer ("Chief") for
his encouragement, guidance, and especially for his enthusiasm,
and lastly, to my wife for her endurance and patience during
critical moments.
iii
TABLE OF CONTENTS
I.
INTRODUCTION
1
II.
MATERIALS AND METHODS
6
A.. Fluorescence Quantitation
1
J..
2.
Equipment
6
1.1 The Microscope
6
1.2 The Photometer
'7
Microfluorometry
B.
III.
Preparation of the Specimen
8
2.2
Focusing the Specimen
9
Application of Method
10
10
3.1 Aldehyde Fixation of
Sarcoma 180 ·( S180) Ce 11 s
10
3.2 Con A Receptor Site Distribution on the Surface
t1embranes of Embryonic Cells
11
Cytochalasin B Treatment of 5180 Tumor
Cells
RESULTS
A.
8
2.1
2.3 Measuring Fluorescence
Intensity of Whole Cells
3.
6
13
14
Fluorescence Quantitation
1.
The Effect of Aldehyde Fixation on
FITC-Con A Binding: A Simple
Illustration of t1icrofluorometry
iv
14
14
2.
B.
The Topographic Distribution of FITCCon A on the Surface of Embryonic Cells
as Determined by Microfluorometry
15
Cytochalasin B Treatment of 5180 Tumor Cells
17
IV.
DISCUSSION
19
V.
BIBLIOGRAPHY
23
VI.
FIGURES AND LEGENDS
27
v
ABSTRACT
THE DISPLAY OF LECTIN RECEPTOR SITES ON THE SURFACES ·
OF TUMOR AND EMBRYONIC CELL POPULATIONS
by
Anthony Neri
~1aster
This study:
of Science in Biology
a) describes a method employing microfluorometry
to measure the fluorescence intensity of cell surface areas labeled
with a fluorescent lectin (fiuoroscein isothiocyanate-conjugated
concanavalin A), b) determines some parameters of binding and
distribution of fluorescent lectins to cell surfaces, c)
shows
that concanavalin A receptor sites appear to be more mobile on
specific migratory embryonic cell populations (sea urchin embryonic
micromeres) as indicated by clustering of the fluorescent labeled
lectin on the cell surface, d)
indicates that cytochalasin B, a
microfilament disrupting drug, affects not only the gross conformation of the plasma memprane but also the distribution of surface
concanavalin A receptor sites of mouse Sarcoma 180 ascites tumor
cells.
vi
.
INTRODUCTION
The cell surface has received much attention both in the fields
. of developmental and cancer biology.
A thorough understanding of its
structure, components, and dynamics would shed more light on numerous
complex cellular processes, and a better understanding of how the cell
interacts with its extracellular envirbnment can provide further
insight into such processes as cell multiplication, morphogenesis,
and malignant invasion (1, 2).
Since the outer cell membrane
~erves
as a direct link with its environment, external stimuli whether chem~cal,
physical, or electrical affect the cell via this organelle.
It is the focus of this report to examine the interaction of
certain cell surface components with a plant lectin (3, 4), concanavalin A (Con A), obtained from the jack bean Canavalia einsformis
(5).
This lectin, among others (6, 7, 8, 9, 10, 11, 12), has the
ability to agglutinate cells of various types by interacting with
their cell surface oligosaccharides (13, 14).
It has been one of the
most widely studied plant lectins, and information regarding its
binding specificities, chemistry, structure, and composition is
available in literature (13, 14, 15, 16).
Briefly, Con A is a
protein that can exist in multimeric forms, depending on pH; at
pH 5.6 or below it exists as a dimer with a molecular weight of
approximately 55,000, consisting of two subunits, each having one
sugar-binding site.
At pH greater than 5.6 the dimers associate
in solution to give a tetramer of approximately 112,000 daltons,
consisting of four subunits, each of the four subunits having one
sugar-binding site (13, 14).
Con A exhibits bihding specificities
1
2
toward oligosaccharides containing
~-0-mannose
or oL-D-glucose
residues (17, 18) which are found to be ptesent on the surface membranes of a variety of normal and transformed cell types (13, 15,
19, 20b).
Con A has been found not only to agglutinate a wide variety of
cells, but also to serve as a mitogen for lymphocytes (21, 22, 23);
restricts cell motility (24), cell growth (25), fertilization (26),
and phagocytosis (27).
In studies involving the dynamics of cell
surface receptors Con A has been found to cause an aggregation of
.receptors
to form clusters and caps in certain cell types (28, 29,
30, 31, 32, 33, 34, 35, 36).
In some cases the ability of these
lectin receptors to be drawn into clusters and caps by Con A molecules has been related to the ability of these tells to be agglutinated by the lectin (13, 15, 19, 20b, 28, 29, 30, 37, 38).
The
treatment of transformed cells with low concentrations of Con A
causes them to agglutinate, whereas their normal counterparts treated
with the same concentrations exhibit little or no agglutination.
This differential agglutination cannot usually be attributed to differences in amount of bound lectin since studies utilizing radioactive
labeled-Con A showed that both the agglutinable and less agglutinable
populations bound relatively the same amounts of lectin (39, 40, 41,
42).
Furthermore topographical distribution of surface bound
ferritin-conjugated Con A in cell aggregates as visualized by electron
microscopy showed that the ferritin-Con A appeared in clusters at
the sites of contact between the agglutinated cells (43).
Treatment
of the aggregates .with the inhibitor .:.4-methyl-D-:mannoside resulted
3
in dispersion of the cells comprising the aggregates.
This result
suggests that ferritin-Con A was responsible for the agglutination.
The Con A-induced localization of the surface receptors to those
sites of intercellular contact has been correlated with the relative
ease by which these receptors can be mobilized to these areas.
Exper-
iments designed to determine. the surface distribution of Con A
receptors of various cell types showed that these receptors are
uniformly distributed if the cells are either aldehyde-fixed prior
to exposure with Con A, or if they are kept at 4°C during or immediately after exposure with Con A (28, 29, 30).
Cells that have this
uniform display pattern also show a decrease in agglutinability.
Therefore it seems that the ability of some cells to agglutinate
depends, in
part~
on factors that allow the receptors to move
laterally in the membrahe in a manner which gives rise to areas of
high recept6r site density.
This implies that the membrane is a
dynamic fluid structure since its components are capable of lateral
diffusion.
Evidence suggesting that membrane components (i.e., proteins)
are capable of lateral movement within the membrane was provided by
Frye and Edidin (44).
They employed the technique of immunofluor-
escence to visualize the intermixing of human and mouse cell surface
antigens on .fused hybrid cells.
Immediately after fusion the human
and mouse antigens remained confined to their respective halves of
the fused cell.
.o
But after some time at 37 C both the human and
mouse antigens were distributed throughout the surface membrane of
the heterokarYon.
Later Singer and Nicolson (45) integrated these,
4
some of their own, and other important observations into the
11
Fluid
Mosaic Model 11 , which has received much attention and is now widely
accepted.
Receptor site mobility, though, is not the only factor involved
in lectin-mediated cell agglutination, but is only one nf a complex
process just beginning to be understood.
Other parameters which
may be involved include cell surface charges, conformation of the
membrane (surface projectibns, microvilli, etc.), the structure and
chemistry of the agglutinating molecules, availability of the
receptors involved, and membrane associated cytoplasmic structures
.
(microtubules and microfilaments) which may restrain the lateral
diffusion of various components through the membrane (13, 20a and b).
The lectin-mediated rearrangement of receptor sites on the
surface:s of different cell types to form local areas of high density,
such as clustering and capping has been reported.
In order to
visualize this process various labels conjugated to the Con A molecule have been utilized.
Labels such as fluorescein isothiocyanate,
ferritin, hemocyanin, and peroxidase coupled to Con A have been the
most widely used.
Although these labels, when conjugated to the
lectin, have provided information regarding receptor site mobility
and distributional differences among cell types, they have not been
utilized in such a manner as to provide quantitative information.
S6me investigators have employed radioisotope labeling of the lectin
molecule to arrive at quantitative binding differences among different
cell types and have obtained reliable results {10, 39, 40, 41).
5
Fluorochrome labels coupled to Con A have been used mainly to
discern distributional differences of Con A receptors displayed by
the surfaces of various cell types (11, 30, 46, 47).
This report
presents (1) a method that enables the investigator to visualize the.
surface display pattern of Con A receptor sites on cells treated with
fluorescein isothiocyanate-Con A (FITC-Con A)',
(2) to simultaneously
obtain quantitative information regarding binding and distribution
of Con A by the use of microfluorometry; and (3) an investigation
of the effects of cytochalasin B, a microfilament disrupting drug
(48), on the surface distribution of Con A receptor sites .
...
MATERIALS AND METHODS
I
I.
A.
Fluorescence Quantitation
1.
Equipment
1.1
The Microscope.
The (eitz Orthoplan microscope
~quipped
with a Leitz fluorescence vertical illuminator was used to provide
fluorescence excitatio~ in incident light.
In this system the
exciting wave length is directed onto the specimen from above through
a series of excitation filters, suppression filters, mirrors, and
ultimately through the objective onto the specimen.
The emitted
Jight, along with non-specific light which reflects back, is captured
by the objectfve and directed through a series rrf built-in suppression
filters.
The unwanted radiation is filtered out and light within a
given wavelength is allowed to pass through.
Inci~ent
illumination
ultimately provides brighter and sharper images by simultaneously
utilizing the objective as a light condenser.
numeri~al
aperture of the objective is
Therefore, as the
increase~,
the specimen will
receive more exciting radiation and hence will increase in fluorescence intensity.
This type of illumination reduces light losses
which can occur through scattering or dispersion, which is possible
with transmitted light excitation.
The microscope also provides
phase contrast illumination of the specimen with or without fluorescence.
Therefore, the specimen can be brought into the plane of
vision by merely utilizing the phase contrast system prior to
photo excitation and hence limit fluorescence fading to a minimum.
The availability and use of this function can be of considerable
6
7
importance when measuring fluorescence intensity.
The fluorochrome in the study presented here was FITC conjugated
to Con
A~
This preparation was obtained from Miles-Veda Laboratories
and was determined not to contain any
tories).
fr~e
FITC (Miles-Veda Labora-
Since the absorptiorr wavelength of this fluorochrome is
approximately 490 nm and the emitted wavelength being approximately
520 nm, the following light filters were used:
excitation filter 5
mm BG12 and a 4 mm BG38 red suppression filter (both in lamp housing);
suppression filters, K495 (built in the vertical illuminator) and
K510 (exchangeable).
The light source used in conjunction with the
filters listed was an XBO 150W, 20V Xenon lamp (Osram, Berlin)
powered by a Leitz power supply (#050230) rated at + 0.1% constant
voltage supply.
A Phaco 40/0.65 objective for phase contrast viewing
of the specimen was used throughout this study except when indicated.
Other objectives such as Phaco 10/0.25 and Phaco ol 100/l .30 can also
be used.
When employing the Phaco ol 100/1.30 it is recommended
that a non-fluorescent immersion oil with a refractive index of
1.515 be used.
Transmitted light for phase contrast observations
was supplied from a 6V, 30W low-voltage lamp powered by a Leitz
variable transformer.
1. 2 · The Photometer.
Mi crofl uorometri c measurements were
performed with the Leitz-MPV photometer system with an adjustable
measuring diaphragm.
The photometer was attached to the Leitz
Orthoplan fluorescence microscope by means of a metal rail
(Aristophot rail, Leitz).
The photomultiplier located in the photo-
meter housing was an EMI 9558 with photocathode type 520.
This type
8
of photomultiplier was used because of its spectral sensitivity for
fluorescence measurements (49,50).
The photomultiplier was
th~n
connected to a Knott NSHM high tension supply unit and to an indicator
(see
*Figur~
1).
The high tension supply unit was set to deliver
l.lKv to the photomultiplier.
For an indicator of fluorescence
intensity a digital picoammeter manufactured by Kethly Instruments,
model 445, was used with a display rate set at 1 per second.
This
type of indicator was chosen because of its sensitivity in reading
small currents (lo- 12 amperes) which are generated by extremely faint
.fluorescent specimens.
In short, the photomultiplier, the high
tension supply unit, and the digital picoammeter comprise the
li9ht measuring system.
2.
Mi crofl uorometry ·
2.1
Preparation of the Specimen. After treating the cell
sample with FITC-Con Ai it is important to remove as much unbound
or free FITC-Con A as possible in order to limit background fluorescence to a minimum.
This was accomplished by washing the treated
cells twice in a suitable buffer.
If aldehyde fixation is incorpor-
ated in the treatment of the specimen, the necessary co.ntrol must be
performed to determine non-specific autofluorescence induced by the
fixative.
We found that Sl80 tumor cells (51) treated only with
phosphate buffered saline containing 1% paraformaldehyde and 0.05%
gluteraldehyde, pH 7.2, contributed as much as 6% fluorescence to the
* Figure 1 was adapted and modified from B8hm &Sprenger,
Histochemie 16, p. 103 (1968).
9
total fluorescence intensity. Therefore, these factors should be
kept in mind during all measurements and appropriate corrections
should be made.
i~
A small volume (25 pl) of the treated cell suspension
placed on a glass slide, covered over with a glass cover slip and
placed on the microscope stage.
2.2 Focusing the Specimen.
A brief diagrammatic representa-
tion of the microfluorometer is shown in Figure 1.
By means of
phase contrast illumination the individual cells are viewed through
the binocular (J), focused, and brought into the plane of vision.
By
this method the cells have not yet been exposed to excitation
radi~tibn
and hence will limit fluorescence fading to a minimum
prior to quantitation of fluorescence intensity.
The binocular
pull-out mirror (E) is moved out cif place and light then reaches
the monocular (K) by reflecting from the mirror (F) located in the
photometer.
The individual cells were refocused again according
to the monocular located at the photometer
housing~
With the mirror
(I) in place, light from the filament lamp (L) passes through
th~
measuring diaphragm (H), reflects from both mirrors (F and G), and
reaches the monocular.
This light will then appear as a bright
field in the center of.the darkef viewing field as seen through the
photome.ter monocular (K).
By carefully adjusting the measuring
diaphragm (H) it is possible to restrict all measurements of
fluorescence intensity to individual cells or to specific areas
within single cells . . In order to maintain the diameter Df the
measuring field constant when measuring fluorescence intensity of
many individual cells, it is important not to readjust the
10
-----·--------~--------~----------------------~
measuring diaphragm (H) between readings.
Thereafter only cells
which conform to the size of the measuring field are used for
fluorescence intensity measurements.
In this way, differences in
fluorescence intensity readings contributed by variations in cell
size can be kept at a minimum.
2.3
Measuring Fluorescence Intensity of Whole Cells.
With
the cell in focus and in the measuring field, fluorescence intensity
measurements can be made.
The phase contrast illumination is turned
off by means of a shutter located beneath the microscope stage
(not shown in Figure 1).
is allowed to pass
Excited light from the Xenon lamp (A)
th~ough
an adjustable field diaphragm (B),
deflected by a beam splitter (D) into the objective (C), and onto
the specimen.
By adjusting the field diaphragm (B) it is possible
to limit excitation to an area that is approximately the same size
as the measuring fieldw
The emitted light reaches the monocular
by passing through the beam splitter (D) and deflecting from the
pull-out mirror (F).
For fluorescence measurements, the pull-out
mirror (F), and the mirror used to change from lamp to measuring
(I) are moved out of place.
When this is done the emitted light
has a direct path to the photomultiplier (M).
The fluorescence
intensity is then displayed by the digital picoammeter indicator
(0) in terms of amperes.
3.
Application of Method
3w1 Aldehyde Fixation of Sarcoma 180 (S180) cells.
180 (S180) cells were
~rown
Sarcoma
in male Swiss white mice for 7 - 14 days.
The animals were sacrificed by cervical dislocation, and the
11
-------·--------------------------~--------~----------------~
peritoneal contents rapidly transferred to Dulbecco's phosphate
buffered
sali~e
pH 7.l (DPBS).
Cells were subjected to 6 washes by
gentle centrifugation in DPBS at 1/5 speed in an International
~
'
clinical centrifuge.
Cells were then resuspended in 10 ml of cold
DPBS, and a viability cell count·using 0.1% trypan blue in DPBS was
performed.
Results from the dye exclusion test showed 99% viability. The
experiment was performed usi~g 5 x 10 6 cell~ per ml for all time
points indicated (See Figure 2}.
.of FITC-Con A
(Miles-Yed~
50
~g
per ml (diluted ih DPBS)
Laboratories) was then added to all
individual cell suspensions and incubated at the appropriate
temperatures.
After incubations, cells were washed twice in 5 ml
of cold DPBS and fixed with DPBS containing 1% paraformaldehyde
and 0.05% glutaraldehyde for 20 minutes at 4° C.
Fixed cells were
again washed twice in 1 ml of cold DPBS and 25pl of the cell
suspensions were transferred onto glass slides and covered with
glass cover slips
(Figure 2).
The effect of prior fixation with
DPBS containing 1% paraformaldehyde and 0.05% glutaraldehyde on
the mean· fluorescence intensity of i ndividua 1 S180 cells is shown
in Figure 3.
Prior to treatment with 50 »g per ml of FITC-Con A,
the cells were exposed to the fixative for 20 minutes at 4° C.
Cells
·were then .washed twice in 10 ml of cold DPBS, incubated for various
times with FITC-Con A at 37° C, washed twice and prepared for
quantitation of fluorescence intensity.
3.2 Con A Receptor Site Distribution on the Surface
Membranes of Embryonic Cells.
Dissociated cells obtai ned at the
12
.--..,.------'--------·-·--------------------~
32/64 cell stage of developing sea urchin embryos (Stronglycentrotus
purpuratus) were used in this study.
The procedures for preparation
of the embryos and the dissociation into single cells have been
previously described (52).
Dissociation of embryos at· this cell
stage (32/64) yields three cell types that are easily distinguished
by size:
micromeres, mesomeres, and macromeres.
Of these, only
the micromeres aremigratory and significantly agglutinable by
Con A (52).
After dissociation into single cells the cell
suspensions were layered on a 5 - 15% Ficoll gradient in order to
remove cell debris, unfertilized eggs or undissociated embryos.
•
The
dissociatedcells were removed from the gradient and washed twice
in calcium and magnesium free sea water (CMF-SW) containing 1%
Ficoll.
An appropriate preparation was then made to include all
cell types at a total concentration of 8 x 10 6 cells/ml of CMF-SW.
One ml of the cell suspension was treated with 750 pg/ml of FITCCon A, incubated at 17° C for 10 minutes, followed with 4%
formaldehyde (prepared in CMF-SvJ containing 1% Ficoll) fixation at
4° C for 20 minutes.
The cells were then washed with CMF-SW
containing 1% Ficoll
and mounted on glass slides.
An individual
ce 11 of either type was then brought into focus by phase contrast
illumination.
This was performed prior to photo excitation in
order to keep fluorescence fading to a minimum.
At this time
diameter measurements were taken and the surface area calculated.
The fluorescence intensity of 10 micromeres and 10 macromeres was
measured and the mean recorded in Table 1.
diaphragm for fluorescence
---·---------
~uantitation
The measuring field
was later adjusted to cover
13
an area of 56.25
2·
p •
Thereafter, the only light reaching the photo-
multiplier was that which was included by the measuring area only.
(Figure 2a and b)
To determine the topographical distribution of
FITC-Con A on the surface of individual cells, the fluorescence
intensity of four distinct areas comprising 56.25 ;/ were measured
(See Table 1 and Figure 3).
B.
Cytochalasin B Treatment of S180 Tumor Cells
Preparation of S180 Cells for Fluorescence Microscopy.
tumor cells
w~re
S180
obtained from the peritoneal cavaties of male Swiss
white mice, washed and viability cell counts performed as in section
The dye exclusion test showed at least 98% viability. The
cells (3.5 x 10 6/ml) were incubated at 37° C for 30 minutes with
3.1.
or without 20 ).lg/ml of cytochalasin B .(CB, Aldrich), prepared in
DPBS containing 1.0% dimethylsulfoxide (DMSO).
FITC-Con A at
100 pg/ml was added to all samples and incubated at 37° C for 10
minutes.
In all cases, cells were washed 3x in DPBS, fixed for
20 minutes with 4% formaldehyde (prepared in DPBS, pH 7.2),
washed twice in DPBS and finally 25 pl transferred to a glass
slide for fluorescence
microscopy~
------------------------~----------------·----------------~
-RESULTS
A.
Fluorescence Quantitation
1.
The Effect of Aldehyde Fixation on FITC-Con A Binding:
Simple Illustration of
A
t-1icrofluorometr~
A simple application of quantitative fluorescence measurements
of mouse tumor cells (S180) treated with FITC-Con A is shown in
Figure 4..
with 50
Results from these data indicate that 5180 cells treated
~g/ml
of FITC-Con A at 4° or 37°C bind approximately the
same amount for up to 20 minutes of incubation.
.
tion
But if the incuba-
time is extended to 30 minutes, the cells incubated at 37oc
bind 30% inore FITC-Con A.
Even though the 30% increase in mean
fluorescence intensity might be a result of the cells internalizing
the labeled lectin, it nevertheless remains clear that quantitative
evaluations can be made.
When comparing cells fixed prior to
treatment with FITC-Con A (Figure 5) to cells fixed after treatment
(Figure 4), the cells fixed prior to exposure with FITC-Con A
had a mean fluorescence intensity 24% less after 20 minutes, and
44% less after 30 minutes.
The
fixativ~
used in this procedure
(see Legend, Figure 5) causes a reduction in the amount of FITCCon A bound by 5180 tumor cells.
Single Cell Variation.
Radioactivity measurements are made
on whole populations of cells.
Important
differ~nces
binding of individual cells in the population
radioactive Con A.
~annat
in Con A
be made using
The method employed here, however, easily enables
measurement of fluorescence intensity of individual cells.
I
.I
14
For
15
example, specific fluorescence intensity readings of ten different·
5180 cell~ exposed to FITC-Con A for 10 minutes, at 4° C, then fixed
for 20 minutes, (see Figure 4) are as follows: 24.5 x 10- 10 ,
22.7
X
10- 10 , 27.]
20 . 4
X
10 -10' 18 . 1 x 10 -10 , 16 .3 •.x 10-10' 24.. 0 x 10 -10 amperes.
X
10- 10 , 19.2
X
10- 10 , 19.6
X
10- 10 ~ 23.0
X
10- 10 ,
Non.:.specifi c fl uorescerice intensity readings due to fixation and
background have been subtracted froni these values.
2.
The Topographic Distribution of
FITC~Con
A on the Surfaces
of Embryonic Cells as Determined by Microfluorometry.
Overall, the sea urchin embryo macromeres had a mean fluorescence
intensity (MFI) significantly higher than the micromeres
(P< .025 by Student•s t test).
to
su~face
When the MFI was calculated according
area (assuming a smooth sphere), the micromeres displayed
greater fluorescence per unit area, however this difference was not
significant at the .05 level.
Although the micromeres were the
highly agglutinable cell type, they did not significantly bind
more FITC-Con A per unit area than the less-agglutinable macromere
population.
The fluorescence intensity of four di sti net areas of 10
individual cells of each type was measured in order to quantitatively
determine any distributional differences of surface bound FITC-Con
The results of these measurements (Tab 1e '1 and Figure 3) indicate
that the micromeres displayed an uneven distribution of receptor
sites as judged by the high fluorescence intensity in one area as
compared to the other three areas measured.
The concentration of
FITC-Con A to one area of the cell, determined both by quantitative
A~
16
measurements and visual observations, indicates a highly clustered or
capp.ed distribution.
The macromeres displayed a relatively even
distribution of fluorescence except for two cells.
to be
~apped
visually
These appeared
when measured quantitatively, but not when observed
(Figure 3). Quantitative measurements can therefore detect
distr~butional
differences of surface bound FITC-Con A that would
otherwise be left undetected by visual examination.
The MFI of all measured areas of individual cells of both
populations showed that a given area on a micromere is 1.35 times
~righter
than the equivalent area of a macromere.
th~ brighte~
However, when
areas or capped regions are omitted from the calculations,
the macromeres have a higher MFI per area measured than micromeres.
This indicates that the capped regions of the micromeres contributed
significantly (52%) to the MFI per area measured.
In contrast,
the contribution made by the brightest areas of the macromeres to
the MFI per area measured is only 16%.
The MFI of capped regions of
micromeres and the brightest areas of noncapped regions of macromeres
(including the areas of the two
CC~;pped
macromeres, Figure 3) indicated
that capped regions of the micromeres had a significantly (P< .005)
greater MFI than macromeres (Table 1).
This suggests that the
micromeres have areas on their surface membranes with a considerably
greater Con A receptor site density than macromeres.
Comparison of
the fluorescence intensity contribution made by the capped areas to
the MFI of 10 cells of each ce 11 type showed that the capped areas
of the micromeres made a contribution of 32.2%. -+ 4.3%,
whereas the
.
contributions made by the macromeres was only
10.8%~
4.7% (Table 1).
17
This difference is significant (P<.025).
B.
Cytochalasin B Treatment of S180 Tumor Cells
When Sl80 cells were treated with CB, then
~xposed
to FITC-Con A,
followed by aldehyde fixation, the surface membrane appeared to
exhibit many large, broad
ruffle~
of FITC-Con A (see Figure 6).
that contained high concentrations
When CB was omitted from the
procedure~
the S180 cells exhibited a more uniform display of fluorescence
without the appearance of any major protrusions or ruffles (Figure 7).
Whether localization of the Con A receptor sites to those ruffles
was mediated by Con A molecules or by CB, was determined by treating
the cells with CB, fixing them, and exposing them to FITC-Con A
(Figure 8).
This experiment indicated that the localization of
the Con A receptors at the ruffles was not as extensive nor as
brightly fluorescent as those in Figure 6.
This suggests that
exposing CB treated cells to FITC-Con A prior to fixation enhances
the localization of receptor sites to those ruffles and simultaneously
causes more pronounced ruffles to be formed.
Figure 9 shows a representative field of S180 cells treated
with CB,
expos~d
to FITC-Con A, followed by formaldehyde fixation.
These cell aggregates showed large amounts of FITC-Con A concentrated
at the sites of cell-to-cell contact.
Experiments which omitted CB
or involved fixation after CB and prior to FITC-Con A treatment,
had fewer cell aggregates.
This suggests that more extensive
ruffling with greater Con A receptor site localization at the ruffles
enhances Con A mediated agglutination.
That the localizSd fluores-
cence observed was due to specific Con A binding and not to non-
18
specific trapping was tested by treating the cells with the competitive binding sugar, o<.-methy-0-mannoside (O.lM).
After treatment with
the competing sugar all fluorescence as visualized by eye disappeared.
This
sugg~sts
that cell surface Con A receptors were being
labeled~
The DMSO that was used to solubilize CB and present at 1.0% concentration throughout this study appeared to have no effect on the gross
conformation of the membrane or on the surface distribution of
FITC-Con A (see Figure 7).
I
I
I·
DISCUSSION
By microfluorometry it is possible to examine the effects of
aldehyde fixation on the binding of FITC-Con A to S180 tumor
cell~.
The purpose of this particular study was only to provide a rather
· simpli.fied application of microfluorometry.
The reliability of
these measurements as compared to radioiosotope methods has been
explored by Killander et ~-, 1970 (53). By using FITC-labeled
antibody and 131 1-labeled antigen, they were able to correlate
fluorescence intensity to radioactivity.
They used F1TC-anti-human
1gM dir.ected against formalini-zed human erythrocytes previously
coated with 131 1-human 1gM (antigeri). The amount of F1TC-anti-human
IgM bound by the erythrocytes was correlated to the amount of
1311-human 1gM bound.
Their results showed that fluorescence inten-
sity (FITC-anti-human 1gM) of single cells decreased as the amount
of protein antigen ( 111 I-human IgM) coated on the surface of the
cells decreased.
1~- t
A strong correlation was found between the amount
of antigen bound to the cell, when measured by radioactivity, and
the fluorescence intensity.
These experiments suggest that quanti-
tative data obtained by measuring fluorescence intensity is as
,,
.
reliable as that which is obtained by measuring radioactivity.
Quantitative evaluations of Con A receptor site distribution on
the surface membranes of embryonic cells showed that the agglutinable
migratory cell population (micromeres) displayed a highly clustered
and capped distribution of Con A receptor sites.
The macromere
population (less-agglutinable cell type) showed a relatively uniform
19
20
distribution of
the~e
receptors.
It was previously demonstrated using
these cell types that the treatment of FITC-Con A-labeled cells with
~-methyl-0-glucoside
(28).
resulted in a loss of visible fluorescence
This indicated that Con A sites were indeed being labeled.
Furthermore, if the cells were fixed with formaldehyde prior to
treatm~nt
with FITC-Con A, all populations displayed a uniform
distribution, suggesting that the Con A molecules were responsible
for drawing the'receptor sites into clusters and caps.
The data obtained here using microfluorometry indicates that
(1) ,the micromeres, being the more agglutinable cell type, do not
bind significantly more FITC-Con A per unit area than the macromeres
and (2) Con A-induced clustering or capping is significantly higher
in micromeres as judged by (a) the higher
va~iation
in fluorescence
intensity of the four areas measured of each individual cell
(Figure 2) and (b) the fluorescence intensity contributions to
the MFI of 10 cells, by the capped areas in micromeres was higher
than that of macromeres (Table 1).
Thus, it appears that Con A
, receptors are more mobile on the surfaces of the migratory micromere
cell population.
Such increased mobility is also observed in
malignant tumor cells (2, 20a and
b)~
CB treatment of S180 cells resulted in a reorganization of Con
A receptor sites to areas exhibiting large, broad ruffles.
Control
experiments omitting CB showed the receptors to be somewhat uniformly
distributed on the surfaces uf these cells.
This gross change in
membrane conformation was paralleled by an enhanced Con A-mediated
cell agglutination as determined by visual observation and by
21
quantitative methods (54).
A high density of Con A molecules at
these ruffled areas may enhance agglutination by providing a greater
number of molecular crossbridging between adjacent cells.
Whether
ruffled areas are due to a disruption of CB-sensitive microfilaments
in this system is not clearly understood.
An extension of this
study involving electron microscopy failed to reveal any such
structures in cells prior to treatment with the drug (Oppenheimer
et
~·,
submitted, 1976).
This· does not preclude their presence
since they may be transient or very short elements difficult to
~etect.
It is clear
t~at
in this system CB affects not only the
gross conformation of the plasma membrane but also the distribution
of certain lectin receptor sites.
Gross changes are also related
to an increased agglutinability of the cells by Con A.
Microfilaments and microtubules comprise the cytoskeleta·l
system of the cell;
These two $tructures are not only associated
with one another but also appear to extend to the plasma membrane
(for review see 20a).
Together they have been implicated in such
processes as cell division (48, 55, 56), cell motility (48, 55, 56,
57,
~8),
morphogenetic movements (48,59), lectin-mediated agglutin-
ation (60), phagocytosis (61), and distribution of cell surface
. lectin receptor sites (46, 62, 63).
It may be that microfilaments
are associated with peripheral membrane components, such as proteins
or·glycoproteins.
This would thus impose restraints on the lateral
mobility of these components through the membrane (64).
Though
microfilaments ~ave been found in close association with the ~lasma
membrane, direct evidence indicating linkage with membrane components
22
r-------------~---~~-----------~
is at present lacking.
However, membrane-associated cytoskeletal
components are not the only factors that may restrain the movements
of components through the plane of the membrane.
These and other
mechanisms which may have an effect have been discussed in detail
by Nicoloson (13, 20a and b, 43).
In summary, this thesis:
(a)
describes a quantitative assay.
to measure fluorescence intensity of cell surface areas labeled with
fluorescent lectins, (b) determines some parameters of binding and
distribution of fluorescent lectins to cell surfaces, (c) showed
.
·that Con· A receptor sites appear to be more mobile on specific
migratory embryonic cell populations and (d) indicated that CB
causes altered distributions of Con A receptor sites on specific
tumor cell types and that these distributional changes appear to
affect Con A-mediated agglutinability of Sarcoma 180 ascites
tumor cells.
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5.
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7.
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---~. ····----··---··-~------·--------·-·--····-·-· -··----·------···-~·--------------
23
. ----
2890 (1970).
··~---·------.......J
24
...,......., .. ,. _
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_...,"~""'_....... ....,-..,.._,..."'_..~- ..... -<.YG.,.• .. ..,_•t.:.> ... ><''I .... _
........-..,.......,_,_.__... .........__,,,_....._..,. __ ,.,....., .. ........ ._. ........
_....,..,_..,.;_ .. ~...,M"WI-•---..
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Robbins, J.C., and Nicol~on, G.L~: Cancer A Comprehensi~e
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·
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·
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26.
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Nature
International
Experimental
Nature New Biology 235: 44 (1972).
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Smith, S.B. ,. and Revel, J-P.:
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Bernhard, vJ.: Cancer Research 34: 230 (1974).
35.
Nicolson, G.L., Yanagimachi, R., and Yanagimachi, H.:
Journal Cell Biology 66: 263' (1975).
36.
Nicolson, G.L.:
Science
Developmental Biology 27:
Experimental Cell
Nature New Biology 239: 193 (1972).
-·--···------.. ---------···~-.---·-··-··----- . -------·---------..,-.·-·---·-----.. ·--~---------·-·-------·---------.J
25
-----~----~----·
--·
---·---·-----------~-------.,------...-,
37.
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(Excerpta Medica, Amsterdam, 1974).
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Nicolson, G.L.: Concanavalin A, Chapter 8 (Plenum Publishing
Corporation, New York, 1975).
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Inoue,
40.
Cline, M.J., and
1~.:
Journal Cell Science.!.!= 197 (1974).
Livingston~
D.C.:. Nature New Biology 232:
155 (1971).
41.
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·44.
Journal Virology 8:
.·
.
-
Nicolson, G.L.; Cont~ol of Proliferation in Animal Cells,
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'(1970).
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46.
de Petris, S.:
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Sachs, L.: Biochimica Biophysica Acta 311:594 (1973).
48.
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49.
Bohm, N., and Sprenger, E.:
50.
Leitz-Hetzlar: Microscope Photometer With Variable
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Roberson, M., and Oppenheimer, S.B.:
Nature 250: 54 (1974).
S.B.:
Research~:
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54.
Oppenheimer, S.B~, and Odancrantz, J.:
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55.
Westermark, B.:
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Experimental Cell Research 82: 341 (1973).
26
..-----·
56.
tarter, S.B.:
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57.
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58.
Dye, F.J.:
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61.
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63.
Paste, G., Papahadjopoulos, D., and Nicolson, G.L.:
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64.
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Journal Cell Biology 62:
Journal
27
'-'
f""'
Figure 1
A brief diagrammatic representation of the microfluorometer.
'~--·~--~------------------~------------------~----------~~
28
M
II IIIII
I
I
L
~----~ ~\
I' '
p
K
<j
-1-I
----~"' \7
<) - - - - I ''\.
<) -----~ E '\.'
I
I
I
u
I
I
0
I
G
A
XENON
LAMP
B
FIELD
DIAPHRAGM
C
PHASE
OBJECTIVE
D
DICHROIC BEAM-SPLITTING
MIRROR
E
PULLOUT MIRROR
F
PULLOUT MIRROR
G
TRIPLE MIRROR
H
MEASURING
FIELD DIAPHRAGM
MOVEABLE Ml RROR TO CHANGE
FROM LAMP TO MEASURING
flA--te---sp.
I
SPECIMEN
N
L E G E N D
H
I
I
J
I
w
J
BINOCULAR
K
MONOCULAR
L
FILAMENT LIGHT SOURCE
M
PHOTOMULTIPLIER
N
HIGH TENSION SUPPLY UNIT
0
INDICATOR UNIT - DIGITAL
PICOAMMETER
C
Figure 1
29
Figure 2
Specific populations of embryonic cells showing the measuring
area; (a)
~lacromere
with measuring area (phase contrast
xl,625), (b) Micromere with measuring area (phase contrast
xl,625).
All measurements of fluorescence intensity have
been represented in Table 1 and Figure 3.
30
Figure 2a
31
Figure 2b
32
------------~-----------------------------------·----------~
Table 1
For measuring the mean fluorescence intensity of 10 cells of
each type, the measuring field diaphragm was adjusted to provide an area which measured the fluorescence .-intensity of only
one cell.
Corrections for fluorescence contributed by the
background during these measurements have been made.
For
measurement of background fluorescence, three empty areas
adjacent to the cell which had been previously quantified were
measured, and the mean subtracted from the tota 1 fluorescence
intensity.
-8
10 amperes.
a
Units of fluorescence intensity are in terms of
·.
Standard error.
An area measured for eac2 individual cell of both
populations was 56.25 ~m •
TABLE
A QUANTITATIVE COMPARISON OF FLUORESCENCE TOPOGRAPHY DISPLAYED ON THE:
SURFACES OF SPECIFIC POPULATIONS OF EMBRYONIC CELLS TREATED WITH F!TC-CON A
CELL TYPE
MEAN SURFACE
AREA (,uti
MICROMERE
1,052.35
:!: 101.91 a
MACROMERE
3,397.97
::!: 307.85
MFI
*
MFI/SURFACE
AREA (}.It)
'MFI/AREA
MEASURED
*
MFI OFAREAS
MEASURED
EXCLUDING
CAP AREAS
MFI OF CAP
AREAS
MEASURED
ONLY
FLUORESCENCE
INTENSITY
CONTR !a!JTION
BY CAP A~EAS
TO MFI
32.2%
::!: il.64
0.058
:!: 0.014
7.23
:!: 1.82
±
3.05
0.44
19.77
:!: 5.66
130.32
± 33.09
0,038
::!: 0.008
5.97
± 0.91
5.00
::!: 0.80
8.93
10.8%
:!: 2.64
:!: 4.7%
55.62
:!: 4.3%
Table 1
w
w
34
-----·--------'-·--------------------,
Figure 3
Fluorescence intensity distribution of four areas measured
on ten individual micromeres (shaded columns} and macromeres (solid columns).
of both types.
Abscissa:
Ordinate:
ten individual cells
fluorescence intensity (%} of
areas measured was derived from (see also Legend to Table 1}:
Intensity for each area measured
Fluorescence intensity of ind1vidua1 cell
~
X 100
35
-.:::1"
~~
N
- ·--u
:/)
()
-u
0
~
0\
00
:::l
·->
·-u
-
r- c
rJ)
<1.)
$...t
<1.)
\0
8
V)
$...t
-.:::1"
0
·-:E
u
~
N
0
c>:J
0
0
rV)
~
p;uns'P.dUI S'P.dJB JO Al!SUdlU! dJUdJSd.IOnt~
M
QJ
s..
::I
Ol
.,..
u. .
36
Figure 4
Each point in the graph represents the mean fluorescence
intensity of ten randomly chosen, single cells, which have
been corrected for autofluorescence (see text).
Corrections
for fluorescence contributed by the background have also
been made.
For measurements of background fluorescence,
three empty areas adjacent to the cell which had been
previouslj quantitated were measured, and the mean subtracted
from the total fluorescence intensity.
in this figure are:
and at 37°C (o-o ).
·-
The data represented
50 ).Jg/ml of FITC-Con A at 4°C (A-,c),
37
60
>~
u;
z
IJJ
~
40
z
IJJ
(,)
z
IJJ
(,)
C/)
IJJ
Q:
0
;:)
...J
u...
z<(
IJJ
~
20
0
10
INCUBATION
20
TIME (min.)
Figure 4
30
Figure 5
The effect of prior fixation with DPBS containing 1% paraformaldehyde and 0.05% glutaraldehyde on the mean fluorescence
intensity of individual S180 cells.
Prior to treatment with
50 JJg/ml of FITC-Con A, the cells were exposed to the
fixative for 20 minutes at 4°C.
Cells were then washed twice
in 10 ml of cold DPBS, incubated for various times with FITCCon A at 37°C, washed twice and prepared for quantitation of
fluorescence intensity, as described in Figure 4.
39
60-
>-
t-
CJ)
z 40-
w
z
t-
w
u
z
w
u
CJ)
w
0::
0
:::>
...1
u..
0------·
20-
0
·------·
----------
10
INCUBATION
30
TIME (min.)
Figure 5
40
Figure 6
Fluorescence micrograph of CB treated (20 )Jg/ml, 30 minutes)
Sl80 cell incubated with FITC-Con A (100 )Jg/ml) and fixed as
described in the text.
Fluorescence is concentrated on what
appear to be large ruffles.
. ---=---------~------------~--------------------------~
41
Figure 6
42
Figure 7
Fluorescence micrograph of contro 1 S180 ce 11 s treated with
buffer (DPBS) containing 1.0% DMSO, followed with FITC-Con A
and
the~
fixed as described in the
te~t.
43
Figure 7
44
Figure 8
.Fluorescence micrograph of Sl80 cell treated with CB
(20 pg/ml, 30 minutes), followed· by fixation, and· then
exposure to FITC-Con A (lOO~g/ml).
l
45
Figure 8
46
Figure 9
Fluorescence micrograph of a representative field of
5180 cells treated with CB
(20~g/ml,
30 minutes),
. exposed to FITC-Con A ( 100 pg/ml), fo 11 O\'Jed by formaldehyde fixation.
The.se cell aggregates exhibited
large amounts of FITC-Con A concentrated at the sites
of the cell-to-cell contact.
~.~~-~----------------------J
47
Figure 9