CALIFORNIA STATE UNIVERSITY, NORTHRIDGE
THE APPLICATION OF PLANT LECTINS TO SPECIFIC QUESTIONS
1\
CONCERNING THE NATURE OF NEOPLASTIC AND EMBRYONIC
CELL SURFACE RECEPTORS
A thesis· submitted in partial satisfaction of the
requirements for the degree of Master of Science in
Biology
. by
Charles F. Sternburg
June, 1978
is approved.::
Ma:rVin H. Cantor, Ph.D.
1varren
California State University, North·rl.dge
ii
ABSTRACT
THE APPLICATION OF PLANT LECTINS TO SPECIFIC QUESTIONS
CONCERNING THE NATURE OF NEOPLASTIC AND
~RYONIC
CELL SURFACE RECEPTORS
by
Charles F. Sternburg
Master of Science in Biology
Lectins are carbohydrate binding proteins considered to be useful
probes in the study of cell surface glycoproteins.
Using microfluoro-
metry, treatment of dissociated sea urchin embryo cells with fluorescein
isothiocyanate-concanava1in A lectin showed differences in the topo-·
graphical distribution of cell surface Con A recept:t."Jr molecules in various embryonic stages.
Capped and clustered distribution of receptor
sites (indicating receptor mobility) appears in 95% ·of early dissociated
embryo celis, but as development proceeds, certain populations of cells
appear exhibiting no lateral mobility of surface receptor sites.
The
results suggest a correlation between cell behavior and surface lectin
receptor site mobility.
Purification of teratoma cell adhesion factor <Jas accomplished by
affinity chromatography with Ricinus communis lec.t.:rn bound covalently
to agarose beads.
Aggregation promoting activity of this factor was
iii
recovered in a single peak when eluted with a D-galactose gradient, and
SDS polyacrylamide gel electrophoresis showed a single band when stained
for protein.
Plant lectins are shown here to be of value in cell surface mapping
and chemical purification of carbohydrate-containing molecules.
iv
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ACKNOWLEDGMENTS
1
I would like to express my sincere thanks to Dr. Warren Furumoto
for his time and consideration; to Dr. Marvin Cantor for his interest,
!guidance, and critical evaluation; to Dr. Harry Highkin for his eni
j
1
couragement and for the use of his facilities; and to Dr. Steve Oppen-
.heimer who befriended me, taught me, and inspired me.
I would also
;like to express my appreciation to James T. Meyer for his friendship
land assistance, and to my wife, Cristina, for her infinite patience and
I!support.
v
TABLE OF CONTENTS
APPROVAL PAGE
ABSTRACT
ii
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ACKNOWLEDGMENTS
. . . . . . . . . . . . . . . . . . . . . . e; . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
v
INTRODUCTION
1
MATERIALS AND METHODS
5
RESULTS
11
DISCUSSION
21
REFERENCES
26
FIGURE 1
15
FIGURE·2
17
FIGURE 3
19
FIGURE 4
FIGURE 5
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19
20
INTRODUCTION
In recent years, the role of the cell surface in differentiation,
morphogenesis, and cell adhesion has been widely investigated.
This
i
work has been greatly facilitated by the discovery and use of lectins,
,carbohydrate binding proteins that agglutinate tumor and embryonic
cells.
The term "lectin" was first proposed by Boyd in 1963, and orig-
inally referred to proteins with the ability to agglutinate erythro:cytes [1].
The first plant lectin to be isolated was wheat germ
'agglutinin (WGA), found by Burger as a contaminant in lipase [ 2].
Burger saw it as a "tumor specific molecule" and thereby attached significance to it.
Subsequently, other lectins were isolated from other
,Plant as well as animal sources, and it was found that each lee tin had
:s
binding specificity for a given monosaccharide [3].
Because of their
sugar binding specificity, lectins have been used to monitor various
carbohydrate groups on the cell surface.
erally referred to as
11
receptors. 11
These surface sugars are gen-
Plant lectins such as Concanavalin A
(Con A) and Ricinus communis agglutinin (RCA) have been used extensively
to study the nature and distribution of surface receptor sites and
!birtding molecules on the surfaces of normal, transformed, and embryonic
:cell types.
These sites may be involved in the control of cellular
imigration and adhesiveness [4,5].
This report deals with the applica-
tion of two different lectins, Concanavalin A and RCA, to questions
,concerning the topographical distribution of surface receptors in the
!
sea urchin, Strongylocentrotus purpuratus, and the purification of a
'cell specific adhesion factor found in the ascites fluid of male 129/J
mice inoculated with teratoma tumor cells.
1
2
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tl1e-courseo.f-aeveToi>men f-or-El:le --sea--urchin·-·-emt>r:Ya-:-·a:t:-Efle--To.:::----
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icell stage, unequal cytoplasmic division gives rise to three different
1
'cell types, easily distinguishable by size.
~re
!
Macromeres and mesomeres
the larger of the three types and they are sessile, or non-
•
~gratory,
in nature.
The smaller of the three types are the micro-
!
meres, and it is this group of small cells that becomes the mesenchyme
:tissue, a group of migratory cells which invaginates into the interior
of the embryo daring gastrulation.
It has been proposed that the
;orderly movement of individual cells during morphogenesis may be due to
;
!differential cell surface properties [6].
Movements of the migratory
:cell types, especially during gastrulation~ exhibit a behavior pattern
!similar to the invasiveness characteristic of malignant cells.
Rober-
:son, et al. and Neri, et al. showed that treatment of dissociated em.bryonic cells from the 16 and 32 cell stage with fluorescent Con A
.causes Con A induced capped distribution of Con A receptor sites of
•certain cells.
It was found that the micromeres were the more agglutin-
able cell type and that Con A induced capping was significantly higher
'in the micromeres as shown by the mean fluorescent intensity of the
:caps· [7, 8].
Non-migratory embryonic cell types of the 16 and 32 cell
stage (macromeres and mesomeres) do not show increased Con A receptor
site mobility, and are not significantly agglutinable with FITC-Con A
·{8].
This information was taken to suggest a direct correlation between
:con A receptor site mobility and cellular migratory activity [7,8,29,9·'.12].
In Part I of this report, agglutinability of all cell types pre.ceding the 16-32 cell stage of sea urchin development are investigated
3
;
!distribution
of Con A receptor sites.
!
Cell adhesion and cell recognition.sometimes appear to be dependent
on aggregation factors.
These are tissue or species specific molecules
!derived from the cell surface or
I'introduced
cells.
suspensi~n
medium that, when re-
to the cell suspension, have the ability to aggregate those
H. V. Wilson first showed cell adhesion and recognition to be
·species speci£ ic in the sponges, Microciona prolifera and Haliclona
i
;occulata [13].
Humphreys and Hoscona later showed this specificity to
"'
;
i
;be due in part to a species specific aggregation factor [14,15].
1
To
date, specific aggregation factors have been isolated from the sponges
mentioned above [16], embryonic chick neural retina [17], embryonic
:chick liver [18], two species of slime mold (Dictyostelium discoideum
'and Polysphondylium pallidum) [19,20], 129/J ascites teratoma [22,23],
and sea urchin sperm [21].
Of these factors, those from the sponges,
slime molds, embryonic chick neural retina, and sea urchin sperm have
been purified.
Part II of this report deals with the use of Ricinus communis agglutinin (RCA), a plant lectin from the castor bean, to isolate and
purify teratoma adhesion factor.
This factor is a cell specific agglu-
;tinating molecule of unknown origin found in minute amounts in the
;ascites fluid of male 129/J mice bearing ascites teratoma tumor [22].
•This is the same molecule that was partially purified by Meyer and
Oppenheimer in 1976 [23].
This molecule has been shown to physically
bind cells together without metabolism [22], and to be dependent on a
functional galactose group for this ability [24].
A method is described
here that takes advantage of this galactose group to fractionate crude
4
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·c:atiimns ___,
I
kvith
covalently bound RCA.
;
RCA is specific for galactose-like residues.
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!
:Reagents and Media
Fluorescein isothiocyanate-concanavalin A (FITC-Con A) and agarose~icinus
communis agglutinin 60 were obtained from Miles-Yeda Ltd.,
:Elkhart, Ind.
Dithiothreitol (DTT, Clel.and's Reagent), N-2-hydroxy-
ethylpiperizine-N'-2-ethanesulfonic acid (HEPES buffer), Deoxyribonu:clease I
(DNAase), (Hydroxymethyl) aminomet.h.ane (Tris buffer), and
iethyleneglycol-bis-(-8-amino-ethyl ether) N,N( tetracetic acid (EGTA)
(were obtained from Sigma Chemical Co., St. Louis, Mo.
Acrylamide,
;sodium dodecyl sulfate (SDS), ammonium persulfate, N ,N ,N' ,N' -tetrabethylethylenediamine (TEMED), bromophenol blue tracking dye, and
;Coomassie Brilliant Blue R-250 (CBB) were obtained from Bio-·Rad Labora....:
:tories, Richmond, Calif.
prepare.d as follows:
Calcium-magnesium·-free sea water (CMF-SliJ) was
2 7. Og NaCl, l. Og Na so , 0. 8g KCl, 0 .18g NaHCO ,
2 4
3
.and lOmg DNAase were dissolved in 1.01 of distilled water, and this was
brought to pH 8.0 \vith Tris buffer.
HEPES-saline solution \vas prepared
'
'by adding 8.176g NaCl, 2.38g HEPES, 0.222g CaC1 , 0.200g MgC1 ·6H 0,
2
2
2
and J.Omg DNAase to 1.0 1 of distilled water.
;with NaOH.
The pH was adjusted to 7.5
Denaturing solution for SDS gel electrophoresis was prepared
by mixing 3,75 ml H,.,O, 0.5 ml TEMED, 0.5 mil ammonium persulfate, and
k
. ;.25 ml 20% SDS.
Preparation of Sea Urchin Cell Suspension
Gametes of the sea urchin,
Stron~yloc.entrotus
·tained by injecting 0 .SH KCl into the body cavity.
purEuratus, were obSperm was kept un-
diluted on ice and the eggs were washed 3X in :iY1illipore-filtered 0.02. H
5
Iris-buffered sea water at pH 8.0 (MF-SW).
eggs were re-suspended in 10 ml MF:...sw.
2.0 ml of gently packed
This suspension was combined
with 10 ml 0.02 M DTT in MF-SW (pH 9.0) and slowly stirred for 4 minutes
at room temperature.
DTT treatment is necessary to prevent the forma-
tion of the fertilization membrane which, if formed, would make cell
separation impossible.
400 ml MF-SW.
with another
After 4 minutes, the suspension is diluted with
The eggs were allowed to settle, and then washed again
!~00
ml MF-SW.
Each 2. 0 ml of DTT treated eggs was sus-
pended in 15-20 ml MF-SW and 10 111 of undiluted sperm were added.
The
suspension was slowly stirred for 45 seconds, poured through a nitex
screen into 400 ml MF:...sw, and immediately transfered to sterile petri
dishes and incubated at l7°C.
When the embryos reached the desired
stage of development, they were gently removed from the petri dishes
with the flat end of a rubber pipette bulb and washed 3X in 0.02 M Trisbuffered calcium-magnesium-free sea water with 1% Ficoll at pH 8.0 (CHFSW).
0.2 ml of 0.01 M EGTA in CMF-SW was added to each 3.0 ml cell sus-
pension to facilitate dissociation of the embryos into single cells by
CMF-SW.
The suspension was incubated at l7°C for 10 minutes and then
gently pipetted lOX with a Pasteur pipette before dilution with 10 ml
CMF-SW.
The dissociated cells were centrifuged at 250 rpm for 3-5 min-
utes in 15 ml conical glass centrifuge tubes on an International Clinicli
Centrifuge with rotor #221, and re-suspended in CMF-SW at a concentrmion
of 2.0 X 10
6
cells/ml.
Microfluorometry
Microfluorometry was done using a Zeiss phase contrast dark field
fluorescence microscope.
The light source was an HBO 200W 12 mercury
7
vapor bulb (Osram, Berlin) powered by a Zeiss power supply.
Trans-
mitted light for phase contrast illumination was from a 6V tungsten
bulb.
FITC-Con A with no free FITC was used as the Fluorochrome.
Its
absorbance wavelengthis 490nm and its emitted wavelength is 520 nm.
A BG-12 excitation filter and a BG-38 red suppression filter were both
mounted in the mercury vapor lamp housing, and a K495 suppression filter
was used in the ocular.
Dissociated cells obtained from the 1-16 cell stages of developing
sea urchins (S. purpuratus) at a concentration of 3 X 10
6
cells/ml of
CMF-SW with 1.0% Ficoll were treated with 750 llg/ml FITC-Con A and incubated at 17.0°C for 10 minutes.
The unbound
FITC~Con
A was washed
from the preparation with CMF-SW and the cells were fixed in 4% formaldehyde in CMF-SW containing 1.0% Ficoll at 4.0°C for 20 minutes.
After
fixation, the cells were washed again in CMF-SW with 1.0% Ficoll and 20
!ll of the sample was mounted on a glass slide with a cover slip and
placed on the microscope.
Each cell preparation was focused with phase
contrast incidence illumination.
The microscope provides phase contrast
illumination of the specimen with or without fluorescence illumination,
so the specimen can be brought into focus without excitation.
keeps fluorescence fading to a minimum.
This
After focusing, each specimen
was viewed under fluorescence illumination, and the entire slide was
scanned.
The various types of surface topographical patterns were
counted and recorded.
Ascites Teratoma Cells
129/J ascites teratoma was acquired from Dr. Leroy Stevens of the
Jackson Laboratory.
The cells were maintained by intraperitoneal pas-
sage in young male 129/ J mice, where they were gro·;,;n for 5-10 days until
8
abdominal bloating was observed.
Mice were killed by cervical disloca-
tion and the contents of the peritoneal cavity were removed into a 50 ml
beaker.
This suspension contains teratoma cells and crude teratoma ad-
hesion .factor (TAF).
The cell suspension was then centrifuged in 15 ml
conical glass centrifuge tubes for 5 minutes at 1020 rpm on an International Clinical Centrifuge with a #221 rotor.
moved, placed in cellulose nitrate
tubes~
The supernatant was re-
and centrifuged again on a
Sorvall RC2-B Superspeed Centrifuge with a type SS-34 rotor at 5000 rpm
for 10 minutes.
The supernatant was removed and stored on ice in a one
dram vial as crude TAF.
Aggregation Assay
The teratoma cells were washed 3X in HEPES-saline buffer pH 7.5
(HS), and suspended in HS at a concentration of 2 X 10
ml of this cell suspension
6
cells/ml.
0.2
was incubated with an equal volume of TAF
at 37°C for 15 minutes in capped 1 dram vials on a gyratory shaker at
66 rpm with a 4-5/8 inch diameter of rotation.
Cells were counted on
an electronic particle counter (Model 112 I.T Electrozone Celloscope,
Particle Data Inc., Elmhurst, Illinois) and the disappearance of single
cells was measured.
Disappearance of single cells is taken as a measure
of the agglutination activity of crude TAF-
Only TAF that was over 80%
active in agglutinating cells was used in these experiments.
Chromatography
1.0 ml of agarose-Ricinus communis agglutinin 60 (Miles-Yeda Ltd.)
with 1.0 mg of protein per 1.0 ml of resin :was poured into a Pasteur
pipette fitted with a small piece of glass wool and washed with 3.0 ml
of IIEPES-saline pH 7.5 at 4.0°C.
0.2 ml of crude TAF was layered onto
9
the top of the agarose bed, and after the sample had entered the column,
was eluted with a 50 ml continuous gradient of 0-0.5 M D-galatose in HS
pH 7.5 at 4.0°C, and collected in 1.0 ml fractions on a Golden Retriever
Pup Model 1100 Fraction Collector (Instrumentation Specialties Co.,
Lincoln, Nebraska).
Elution was by gravity in all experiments, and the
flow rate was approximately 30 ml per hour with a slight increase at
high galactose concentrations.
All fractions were dialyzed against9.0 1
of HS pH 7.5 at 4.0°C for 12 hours to remove galactose and protein elution profiles were determined on a Beckman DB-G grating spectrophotometer.
Absorbance was read at 280 nm using quartz spectrophotometer
cells (Lightpath Cells, Inc., St. Louis, Mo.), and aggregation promoting
activity of the fractions was tested at 50% vol/vol.
Protein determina-
tion was by Lowry method [25].
SDS Gel Electrophoresis
SDS gels were prepared using a procedure outlined by Lammli [26].
5% polyacrylamide gels with 0.1% sodium dodecyl sulfate were poured to
a height of 10 em in precision bore 5 mm X 12.5 em glass tubes (Bio-Rad,
Richmond, Calif.).
Sample denaturing was as follows:
10 ]Jl of dena-
turing solution, 5 ]Jl of 2 mercaptoethanol, 10 ]Jl of tracking dye, 75
]Jl of protein sample, and one crystal of sucrose were mixed and heated
to 100°C for 2 minutes in a small test tube.
The denatured protein
sample was layered onto the gels and electrophoresis was carried out at
3 mA/gel on a d.c. power supply (Buchler Instruments, Chicago, Ill.) at
room temperature with an electrode buffer of 0.25 M Tris-0.192 M glycine
and 0.1% SDS.
Current was applied until the tracking dye reached the
bottom of the gels and they were removed and stained in a solution of
0.05% CBB, 25.0% isopropyl alcohol, 10.0% acetic acid, and 65.0% H o for
2
10
6 hours.
Destaining was carried out in a solution of 0.0025% CBB, 10.0%
isopropyl alcohol, 10.0% acetic acid, and 80.0% H o for 20-30 hours and
2
then in 7.0% acetic acid.
RESULTS
Part I: Distribution of FITc .... con A Receptor Sites in Early Sea Urchin
Embryos
Treatment of dissociated sea urchin embryonic cells with FITC-Con A
showed differences in topographical distribution of Con A receptors in
the different stages of development tested.
Unfertilized eggs and fer-
tilized eggs up to two hours after fertilization showed uniform fluorescence in over 95% of all samples.
receptor sites was seen.
No capping or clustering of Con A
After two hours of development, fertilized
eggs undergo first cleavage.
After cleavage, the dissociated 2-cell
stage showed clustering of fluorescent sites in 95% of the cells.
Capped
distribution of sites in the 2-cell stage was infrequent, but 95% of the
dissociated 4-cell stage blastomeres showed distinct caps (figs. 1 and
2).
In these early stages of development, all the cells seem to be
agglutinable with FITC Con A and show capped or clustered distribution
of receptor sites.
As development progresses, this capping ability be-
comes restricted to fewer cells in the population.
At the 8-cell stage,
the cells are all morphologically identical yet capping is seen in only
about 50% of the cells.
rescence.
The rest of the cells have very uniform fluo-
In dissociated preparations from the 16-cell stage of devel-
opment, only 24% capping is seen.
This is consistent with results re-
ported by Roberson, et al. showing only the micromeres to be agglutinable with FITC-Con A in the 16 and
32 cell stage.
Hapten inhibition studies were done using a-methyl-D-mannoside.
Incubation with this monosaccharide resulted in loss of visible fluo' rescence in the cell preparations tested, indicating that the lectin was
indeed bound to the surface of the cell and not internalized.
11
12
Cells from all stages, when fixed first for 20 minutes at 4°C with
4% formaldehyde in CMF-SW and then treated with FITC-Con A showed uniform fluorescence in all cell types.
Part II:
Purification of TAF
Elution of agarose-RCA 60 columns with the galactose gradient produced similar protein elution profiles. in four separate experiments.
When crude active TAF was filtered through the resin, six protein peaks
were consistently obtained (fig. 3).
In peak #7, which came off of the
column at .45 M galactose concentration, aggregation promoting activity
in two.experiments was 90% when assayed at 50% vol/vol with cells.
In
a third experiment, it was 58%, and in a fourth it was 50%.
In order to insure that the aggregation promoting activity was not.
due to release of the lectin from the resin, a 2.0 ml column, identical
to those used in the four experiments, was run with the galactose gradient but without the crude TAF sample (fig. 4).
Absorbance at 280 nm
showed no significant amount of protein in any of the fractions, and aggregation promoting activity was below 5% in all fractions.
In order to determine whether or not there was an increase in specific acti-vity, unit activity was defined as the amount of protein required to aggregate 50% of the cells in a 0.2 ml suspension of 2 X 10
cells/ml in 15 minutes at 37°C.
6
It was found that the specific activity
of the aggregation promoting fractions was 4.52 U/mg protein, the crude
TAF having a specific activity of 1.0 U/mg.
17.6 mg/ml of crude TAF was
needed to cause· 50% aggregation, while in the active fractions, only 3.9
mg/ml was required.
13
SDS polyacrylamide gel electrophoresis was done with crude TAF and
on all active fractions from the column runs.
All gels were run under
identical conditions and stained for protein with CBB.
Crude TAF had
10-12 distinct bands, while all active fractions showed a single band
(fig. 7).
14
Fig. 1
Occurrence of capped and clustered distribution of FITC-Con A receptor sites in early embryonic populations of dissociated cells from
'the sea urchin, S. purpuratus.
Ordinate
Percent of cells found with capped or clustered
surface distribution of FITC-Con A receptor sites
Abscissa
Embryonic development (1-16 cell stag;?.)
Bars
Standard deviation (6 separate experiments)
15
100%
80%
60%
40%
20%
0
1
4
2
Fig. 1
8
16
16
Fig. 2
Occurrence of capped distribution of FITC-Gon A receptor sites in
dissociated populations of early embryonic cells from the sea urchin,
S. purpuratus.
Ordinate
Percent of cells found with capped distribution of
surface FITC-Con A receptor sites
Abscissa
Embryonic development (1-16 cell stage)
Bars
Standard deviation (6 separate experiments)
17
1
2
4
Fig. 2
8
16
18
Fig. 3
Fractionation of crude ascites fluid using agarose-RCA 60 affinity
chromatography and the aggregation promoting activity of each column
fraction.
Ordinate (left)
Absorbance at 280nm
Ordinate (right)
Percent aggregation
Abscissa
Fraction It (ml.)
Absorbance profile for protein (------.) was determined by measuring
absorbance at 280 nm.
Aggregation promoting activity (- - -) was de-
termined for each fraction at 50% vol/vol with a single cell suspension
of 2 X 10
6
cells/ml.
Column dimensions, Smm X Scm.
Flm..Y rate, 30 ml/hr.
Elution buffer, continuous gradient OM-O.SM galactose in HS, pH 7.5.
Fig. 4
Agarose-RCA 60 column with galactose gradient
crude TAF.
Ordinate (left)
Absorbance 280nm
Ordinate (right)
Percent aggregation
Abscissa
Fraction If rol.
Protein absorbance profile. (
Percent aggregation (-
- -
.-)
)
(0-.5~D
run without
19
.30
1
100%
'
~
II
",:
75%
II'I
.20
I l
50%
I I
I 71
I I
.10
25%
6I
.,/',
0
5
10
15
20
25
30
35
40
45
0
Fig. 3
.30
I
100%
75%
.20
50%
.10
25%
0
Fig. 4
20
a
b
c
d
e
Fig. 5
SDS polyacrylamide gels showing (a) crude ascites fluid and
(b,c,d,e ) column fractions active in promo ting aggregation of single
cell teratoma suspension.
stain.
Gels were stained for protein with CBB
DISCUSSION
A.
Surface Lectin Receptor Sites on Embryonic Cells
Moscona showed embryonic chick neural retina to be agglutinable
with lectins [4], and Moscona and Kleinschuster reported that this agglutinability differs with cell age.
tend to lose agglutinability [27].
Cells from older embryonic tissues
Krach, et al. found a decrease in
agglutinability in older embryonic stages in the sea urchin, S. purpuratus, cells from 7-day-old embryos being
from 1-day-old embryos [28].
less agglutinable than cells
Agglutinability of earlier embryonic sea
urchin cells was also tested in this lab, and it was found that at the
32-64 cell stage of development micromeres were significantly more agglutinable with Con-A than macromeres or mesomeres [28].
It was also
shown that micromeres, being the more agglutinable cell type, do not
bind significantly more FITC-Con A per unit of surface area than the
macromeres or mesomeres [8], so agglutination in this system does not
depend on the amount of lectin bound, but actually depends on other
factors such as lateral mobility of receptor sites through the cell
membrane.
In this study, we investigated the earliest stages of urchin development in an attempt to discover just when capping or clustering of
receptor sites with FITC-Con A first appears.
We have found that capped
and clustered distribution of sites first appears in the dissociated 2cell stage immediately following first cleavage at about 2 hours after
fertilization.
Normally, after fertilization in the sea urchin, a fertilization
membrane is released from the surface of the egg, surrounding and
21
22
protecting it until the hatching stage.
In order to dissociate embryos
into single cells, it is necessary to prevent this fertilization membrane from forming by addition of DTT.
left covering the zygote.
Thus the vitelline membrane is
It is possible that this vestigial membrane
could act on the cell surface to prevent any lateral mobility of membnme
components that could, under more natural conditions, produce a capping
effect.
To clarify this point, further work is needed.
A mechanical
method of removing the fertilization membrane could reveal whether or
not we are dealing with artifact of DTT treatment in the fertilized and
unfertilized egg.
With first cleavage, clustered distribution of receptor sites is
seen in 95% of dissociated cell samples.
It is possible that this
clustering is restricted to the areas of new membrane synthesis that
appear between the cells at first cleavage.
This new membrane would be
the first on the embryo to be .free of the vestigial vitelline layer.
As the embryo grows and more new membrane is formed, localization of
FITC-Con A receptors into caps becomes apparent in over 90% of the 4cell stage dissociated cells.
As development progresses to the 8-cell
stage, and then to the 16-cell stage, the outer cells of the embryo are
becoming more sessile in nature.
As cells differentiate into macromeres
and mesomeres, their .fates are determined as non-migratory cell types
which have been shown previously not to be agglutinable with FITC-Con A
[7,8].
Those cells that differentiate into micromeres and later invagi-
nate into the interior of the embryo have been shown to retain their agglutinability with FITC-Con A [7 ,8].
Figures 1 and 2 suggest a locali-
zation of membrane fluidity that proceeds gradually through the 4, 8,
16, and 32-cell stage until it is finally found only on micromere
23
populations.
Capped and clustered distribution of FITC-Con A receptor
sites (a visual representation of membrane fluidity) first appears in
all early dissociated embryonic cells, but as development proceeds,
sessile, or non-migratory cells appear with no lateral mobility of surface receptors as indicated by uniform FITC-Con A binding.
B.
Purification of Teratoma Cell Adhesion Factor
Glycoproteins, glycolipids, and polysaccharides have been suggested
as possible mediators of intercellular adhesion [5,22-24,30].
Evidence
gathered in this laboratory so far suggests that TAF is a natural molecule of unknown origin, found together with single cells in the ascites
fluid of mice, that has the ability to agglutinate cells non-metabolically in the presence of calcium [22].
Ultrafiltration experiments sug-
gest that the molecular weight is greater than 300,000 D [23], and gel
filtration with Sepharose 6B shows it to be approximately 1,000,000 D
[22].
Hapten inhibition tests suggest that this protein may be the
structural carrier for a D-galactose-containing carbohydrate functionally involved in intercellular aggregation promoting activity [24].
In past work, the inconsistent nature of TAF itself has been a
problem in attempts to purify it.
Mouse ascites fluid is, at this time,
a mass of undefined substances, and cell aggregation promoting activity
is not consistent from sample to sample when removed from the mouse.
Despite efforts to determine a pattern to its periods of activity, no
such cyclic nature has been shown to exist.
Activity can disappear for
months at a time and suddenly reappear at any time.
In the course of
this experiment, it was noticed that TAF which had dramatic aggregation
'
promoting activity on cells from one mouse could have little or no effeet on the same cells taken from a different mouse.
From this it is
24
suggested that agglutinability with active TAF is somehow cell cycle
dependent.
Proteins on the cell surface may differ at different stages
in the cells' development and the cells may not always be agglutinable
with TAF.
Ascites fluid varies widely in viscosity from sample to sample, and
it was often found to be too viscous to run on the agarose-RCA columns.
If the ascites fluid was too thick, the columns would plug and the flow
stopped as soon as the sample entered the resin bed.
Active TAF with a
low enough viscosity to fall through the column was extremely rare, and,
even when it was available, occurred in very small amounts.
Meyer and Oppenheimer showed TAF to have a neutral or cationic component that was separable from an anionic component on DEAE-cellulose
[23].
It was not determined whether or not one of these components was
a subunitof the other.
Affinity chromatography with agarose-RCA shows
aggregation promoting activity recovered in a single protein peak, and
SDS gel electrophoresis shows a single faint protein band for all active
samples tested (fig. 5).
Figure 4 shows that recovered activity was not
due to release of the lectin from the resin, so this new data would suggest that the components separated on DEAE-cellulose actually exist together, and are separated by the ionic gradient.
Because of the unavailability of usable sample, PAS staining [31]
to test for carbohydrate was not done.
Therefore, it remains to be de-
termined whether or not the single band seen on the gels is a glycoprotein.
The evidence gathered to date, however, suggests that it is.
Gel filtration with Sephadex GlOO or GlSO may l:e a possible method
of removing much of the inactive component from the crude TAF before
placing it on agarose-RCA, but this method also
ren~.i.ins
to be tested.
25
Moscona has postulated a ilcell-ligand" hypothesis [32] which suggests that cell recognition and adhesion are controlled by the interaction of cell surface cementing components with cell surface receptor
sites.
These cementing components are referred to as cell ligands, and
experiments suggest that they consist of glycoprotein complexes (22-24].
The degree of complimentarity of cell ligands and surface receptor sites
determines the degree of adhesiveness between two adjacent cells.
TAF
could possibly be such a cell ligand which, after removal from the cell
surface, can cause cell aggregation promoting activity when reintroduced
to a suspension of single cells.
REFERENCES
1.
Boyd, W. C., Vox Sanguinis, 8:
1-32 (1963).
2.
Burger, M. M., and Goldberg, A. R., Biochemistry 57:
3.
Sharon, N., and Lis, H., Science 177:
4.
Moscona, A. A., Science 171:
5.
Oppenheimer, S. B., Edidin, M., Orr, C. W., and Roseman, S., Proc.
Natl. Acad. Sci., U.S.A., 63: 1395-1402 (1969).
6.
Townes, P. L., and Holtfreter, J., Exp. Zool., 128:
7.
Roberson, M., Neri, A., and Oppenheimer, S. B., Science, 189:
640 (1975).
8.
Neri, A., Roberson, M., Connolly, D., and Oppenheimer, S. B., Nature,
258: 342-344 (1975).
9.
Noonan, K. D., and Burger, M. M., J. Cell Bioi., 59:
359-363 (1967).
949-957 (1972).
905-907 (1971).
53-120 (1955).
639-
134 (1973).
10.
Nicolson, G. L., Concanavalin A, (edited by Chowdhury and Weiss)
153-172 (1975).
11.
Nicolson, G. L.' and Yanagimachi, R.' Science, 184:
12.
Inbar, M.' Huet, c. , Oseroff, A. R.' Ben-Bassat, H.' and Sachs,
Biochem. Biophys. Acta., 311: 594-599 (1973).
13.
Wilson, H. v.' J. Exp. Zool., 5:
14.
Humphreys, T., Dev. Biol., 8:
15.
Moscona, A. A., Proc. Natl. Acad. Sci., U.S.A., 49 (1963).
16.
Henkart, P., and Humphreys, T., Biochemistry, 12:
17.
Lilien, J. E., and Moscona, A. A., Science, 157:
18.
Kuroda, Y., Exp. Cell Res.; 49:
19.
Rosen, S.D., Kafka, J. A., Simpson, D. L., and Barondes, S. H.,
Proc. Nat. Acad. Sci., U.S.A., 70: 2554 (1973).
20.
Simpson, D. L., Rosen, S.D., and Barondes, S. H., Biochem. Biophys.
Acta., 412: 109-119 (1975).
21.
Vacquier, V., and May, G., Proc. Nat1. Acad. Sci., U.S.A., 74:
2456-2460 (1977).
1294-1296 (1974).
c.'
245-258 (1907).
27 (1963).
3045 (1973).
70 (1967).
629 (1968).
26
22.
Oppenheimer, S. B., and Humphreys, T., Nature, 232:
23.
Meyer, J. T., and Oppenheimer, S. B., Exper. Cell Res., 102:
364 (1976).
24.
Oppenheimer, S. B., Exp. Cell Res., 92:
25.
Lowry, 0. H., Rosebrough, N.J., Farr, A. L., and Randal, R. J.,
J. Biol. Chern., 193: 265 (1951).
26.
Lammli, U., Nature, 227:
27.
Kleinschuster, S .. , and Moscona, A. A., Exp. Cell Res., 70:
(1972) .
28.
Krach, S. W., Green, A., Nicolson, G. L., and Oppenheimer, S. B.,
Exp. Cell Res., 84: 191-198 (1974).
29.
Roberson, M., and Oppenheimer, S. B., Exp. Cell Res., 91:
(1975).
30.
Oppenheimer, S. B., Exp. Cell Res., 77:
31.
Kapitany, R. A., and Zebrowski, E. J., Anal. Biochem., 56:
(1973).
32.
Moscona, A. A., Devel. Biol., 18:
33.
Cuatrecasas, P., J. Biol. Chern., 245 #12:
34.
Ki1lander, D., Levin, A., Masaharu, I., and Klein, I., Immunology,
19: 151 (1970).
35.
Cochran, C., and Englemann, F., Biol. Bull., 148:
36.
Neri, A., Roberson, M., and Oppenheimer, S. B., in Concanavalin A
as a Tool (edited by Bittiger and Schnebli) Hiley, pp. 221-228.
122~126
125 (1971).
359-
(1975).
680 (1970).
27
397
263-268
175 (1973).
361
250 (1968).
3059-3065 (1970).
393-401 (1975).