CALIFORNIA STATE UNIVERSITY, NORTHRIDGE
A FINE STRUCTURAL
AN~~YSIS
OF SURFACE
~'
INTERACTIONS OF AGGLUTINATED SARCOMA,l80 CELLS
A thesis submitted in partial satisfaction of the
requirements for the degree of Master of Science in
Biology
by
Rudolf Carl Kuppers, Jr.
January, 1976
The thesis of Rudolf Carl Kuppers, Jr. is approved:
California State University, Northridge
December, 1975
-__________.JI
ii
,------------------------------------------------------------- ----------------- ------------------------------------------ ----l
I
I
ACKNOWLEDGEMENT
I would like to acknowledge my committee for their patience before
and during the writing of this thesis. In particular, Dr. Edward
Pollock for his continued inspiration and friendship; Dr. Steven
Oppenheimer for his unceasing energy; and Dr. Marvin Cantor for his
continued friendship. In addition, I \vould like to thank Hr. Geoff
Brennerman, Miss Miriam Burton, and Miss Frederica Witzke for their
help in this \vork.
iii
,~
--- .. ··-·-·--- ·-- - - --·-·--
~---------- ------~ --------------~-----~---------------------1
I
iii
I
II
iv
I1
Table of Contents
!I Acknmv-ledgment
.I
I
I
I
Table of Contents
Abstract
v
Introduction
l
Methods and Materials
7
Maintenance and preparation of Sarcoma 180
7
Viability Test
7
Electron Micros6opy
7
Lanthanium Oxide Staining
8
Ruthenium Red Staining
9
Concanavalin A Agglutination
9
Factor Agglutination
10
Manganese Ion Agglutination
11
12
Results
Fine Structure of Sarcoma 180
12
Concanavalin A Agglutination
15
Factor Agglutination
19
Manganese Ion Agglutination
21
Discussion
24
:Bibliography
37
Table
44
Plates
45
;
I
I
L_____ _
--- -- ... ___ _J
iv
r·--~-
. --- - - - - - - - - - - - -
-------------~--------
-------------------------.---------- - - - - - - - - - - - - - --------
--~~~ ~-
-
~-~
II
ABSTRACT
I
l
AN ULTRASTRUCTURAL ANALYSIS OF THE FINE STRUCTURE
AND SURFACE INTERACTIONS OF AGGLUTINATED SARCOMA 180 CELLS
by
Rudolf Carl Kuppers, Jr.
Master of Science in Biology
January, 1976
Sarcoma 180, a mouse ascites tumor, was examined ultrastructurally using transmission electron microscopy.
Its general fine struc-
ture, and the cell surface interactions occurring as a result of
agglutination were examined.
Three types of agglutinins were used,
Concanavalin A (Con A), an ascites "factor", and manganese ion.
Ultrastructurally, Sarcoma 180 had the features of many tumors:
A large heterochromatic, multilobate nucleus with numerous nucleoli,
reduced amounts of endoplasmic reticulum, and numerous microvilli on
the cell surface.
The mitochondria were somewhat pleomorphic.
Large
lipid droplets were seen in addition to several other large, darkly
stained bodies in the cytoplasm.
Cell surface interactions between
untreated cells were not commonly seen.
Three surface interactions were seen after agglutination:
1)
Microvillous associations characterized by the interaction of villi
v
r~-------···---·------------- --~--·--·
-------------·----------·---------------.. ----------------------1
I
!
I
I
I
!
I
!I
I
I
I
with adjacent cell surfaces; 2) intermediate associations in which
.,
adjacent cell membranes were approximately parallel, crosslinked by
I
patches of lightly stained material, and separated by greater than
I
!
0
I
I
I
.
I
lOOA; and 3) close association in which the cells were separated by
1
I
0
0
less than lOOA, commonly 20A, and resembled gap junctions.
All three types of associations were seen after Con A agglutination.
The intermediate association may represent actual cross-
linking by Con A aggregates.
The close associations, it was argued,
resulted from interactions between cell surface components.
Micro-
villous associations were most commonly seen, though their significance was not clear.
The ascites "factor" at high concentration induced pinocytosis
and vacuolization.
Agglutination resulted from large aggregates of
flocculent material crosslinking the cells.
At lower concentration,
intermediate associations were seen, small aggregates of "factor"
possibly crosslinked the cells together.
Manganese ion produced a very rapid agglutination mediated
totally by villous interactions.
Points of interaction were very
tight, with no obvious gap bet·ween membranes.
It was thought that
ionic bridging, or denaturation of surface components was involved.
·-
L ..,--.--·-
vi
l
--~~--~-·-J
Introduction
In recent years several lines of investigation have shown that
the surfaces of transformed and malignant cells are different from
those 6f "normal'' cells.
These differences include biochemical al-
terations, changes in cell-cell adhesion and of cell surface charge,
and agglutinability,
Gasic and Gasic (1963, 1962) provide the first convincing evi-
I
deuce of a carbohydrate-rich layer external to the plasma membrane of '
mammalian cells.
the
1
This cell coat was conceptualized for all cells as
glycocalyx 1 by Bennett (1963).
The work of several investiga-
tors seems to confirm that the cell coat. of neoplastic cells differs
from that found on normal cells.
Both increases and decreases of
various constituents in the glycocalyx have been reported (Buck, et
al., 1970; Brady, et al., 1969; Martinez-Paloma, et al., 1969;
Meezan, et al., 1969; Mora, et al., 1969; Wu, et al., 1969; Morgan,
1968; Ohta, et al., 1968; Kraemer, 1966; Purdom, et al., 1958).
Coman (1944) demonstrated a general decrease in adhesiveness
bet\veen epithelial tumor cells in comparison with normal epithelium.
This reduction in adhesion has been postulated to account for the
I
change in "social" behavior demonstrated by transformed cells .. Trans-\
I
formed cells show a loss of contact inhibition of growth and mobility!
'
(Dulbecco, 1970; Abercrombie and Ambrose, 1962; Vogt and Dulbecco,
. - - ···-· ,-~---·-- ------. ---- --· ~~--- --·--
---- --- .. --- --·.
--~----------------------------·-.
1
-·· ---- -------------- --· - -- ------------------
-----~-·--· --~-----·.
I
-----l
r-·----------------· ----·------···- - - - --·----------·-· ----------------·-· . --------------·---.. ------------------------.
I
This change in adhesion is not a simple change reflected in
i 1962).
!
Ian overall decrease
II specifically
in adhesion to other cells and substrates, but
cell-cell adhesion.
Indeed, it can be shown that some
I
i
transformed cells stick to other cell types and substrates quite well;
1 for example glass, plastic, or millipore filters (Ambrose, 1967).
I
I This
change in adhesion may be the cause of metastatic behavior in
I
I
tumors ...
!
It has been suggested that the change in cell surface components
Ii leads to a greater number of negative charges and therefore greater
Irepulsion between tumor cells. This is coupled with decreased adhesI
ion (Abercrombie and Ambrose, 1962).
1
I (1958) have shown that
Isublines of MCIM mouse
! so 1 id
Purdom, Ambrose, and Klein
the potential at the cell surface of different
sarcoma increased as they progressed from the
tumor to the ascites form.
\Vhile these studies are
i
I
I adhesion, intensive research
lI mechanism(s)
suggestive in the understanding of cell
has failed to reveal the nature of the
involved in adhesion and their relationship to tumor be-
l
lhavior and growth.
!
One of the most exciting areas in membrane research in recent
I
!years has involved the use of plant lectins or agglutinins to probe
l
i the structure of the cell surface. Lectins are proteins or glycoI
l
'
j
proteins with highly specific, differential saccharide-binding capa-
I
I
I
II
i
l' bilities.
i
f
Exampl~s
are Concanavalin A (Con A) which binds
a-D-
I
imannopyranosyl, a-D-glucopyranosyl and sterically related terminal
I
l
I
isugar residues (Agrmval and Goldstein, 1967), and wheat germ agglutinid
...... ----------·•"""- ··---···------ ........J
3
,---------------·--------------· ---------·-····-----------------------------·--------------"---------··--- ------1
!
!
I
II (WGA)
which binds N-acetyl-D-glucosamine and similar groups (Burger
I
!and Goldberg, 1967).
Il creased
I
I cells
The work of Burger and Goldberg, shmving in-
agglutinability by transformed cells as compared with normal
-
using WGA, provided a stimulus for the use of lectins in the
!
!vestigation of tumor cell surfaces.
I
in- I
In general, it has been shown
I
that transformed cells agglutinate with much lmver concentrations of
I lectin than needed for the corresponding
! al., 1971; Inbar and Sachs, 1969; Burger
normal cell line (Sela, et
and Goldberg, 1967).
I
I
It was originally thought that the increased agglutinability
I demonstrated by
I bers of, or the
transformed cells with Con A was due to increased num-1
i
unmasking of "cryptic", Con-A-binding sites.
A large
I
j amount of evidence now suggests these interpretations are not correct
i
I and,
in fact, nearly the same amount of Con A \vill bind to normal
'
i
! cells as will bind transformed cells of a particular cell line (Arndt!
!
IJavin and Berg,
1971; Cline and Livingston, 1971; Ozanne and Sambrook,
I
11971; Sela, et al., 1971).
!
I
The relative agglutinability of a cell has now been correlated
with the topological distribution of Con A bound to the cell surface.
l In
normal cells the Con A binding sites appear to be randomly dis-
! persed
\vhile these sites are clustered on the transformed cell (Nicol-
I
I son,
I
1971).
The concentration of binding sites in these clusters ap-
,
11
i
: pears to promote crosslinking betHeen cells by the tetravalent Con A
re~·ulting
in cell agglutination.
Apparently, Con A binding sites ·
have an increased freedom of movement in transformed cells allowing
these sites to cluster under the influence of Con A
............ __ .....
. ............... - -- ............................ ------ ·- .... ----. --
.
.
(Nicol~on,
-----· --.------- -
1973;
1
I
!i
i
.. -- .. --· ....... --_I
4
Rosenblith, et al., 1973).
Agglutination can be induced by other mechanisms as well.
For
example, there have been some studies in which poorly defined "factors" from the ascites fluid of tumor bearing mice have been used to
bring about agglutination.
In the case of a mouse teratoma, this
factor was shown to be possibly a glycoprotein and to possess some
I
gree of specificity for the cell line from which it was isolated
(Oppenheimer and
Humphreys~
1971).
That these "factors" have any im-
portance in cell-cell adhesion is unknown.
Specific metal ions such as manganese and lanthanium have been
shown to increase cellular adhesion to substrates and to cause cell
agglutination.
!
I
de-l
But again, the mechanism of their action or its bio-
logical significance has yet to be demonstrated (Rabinovitch and De
I
I
I
l
I
.
Stefano, 1973.; Levinson, et al., 1972; Oppenheimer, personal communi- 1!!
cation).
There are several structural specializations which can occur
normally between cells and hence are implicated in cell adhesion.
These associations are called junctional complexes.
They involve in-
tercellular, plasma membrane, and cytoplasmic specializations.
These
II
!
!
structures are found universally throughout the animal kingdom. Hence,
I
they appear to have an important role in cell-cell associations,
I
though what this role is has not been completely elucidated (McNutt
and Weinstein, 1973).
Junctional complexes are classified into four major types:
1) the!
l
zo
~~-~~ ·- ~~~_:_r_~~~---~2- __:~~~~~-~-~~-~-=~~~--_?-~--~t!Xt~ ~-'----~ ~---~~-=--? =-~~?. s ~~-= -'---~~-~---~
5
4)
the tight junction or zonula occludens (see McNutt and Weinstein,
1973 for a complete review).
There is some evidence to suggest these
structures are involved in adhesion between cells.
The use of hy-
pertonic media (Brightman and Reese, 1969), or the incubation of
cardiac muscle in Ca++-free media (Muir, 1967), reveals tight june-
l
!
tions, gap junctions and/or desmosomes which remain after these treat-1
ments, keeping the cells attached.
The gap junction has been impli-
I
cated as· the pathway for electrical coupling and the passage way of
small molecules between cells.
the
11
It has been suggested that some of
social 11 behavior of cells may be mediated by communication
through this type of junctional complex.
(Bennett, 1973; Lowenstein,
1973).
In general, a relationship appears to exist between the number
of junctional complexes and cell normalcy, tumor cells having fe>ver
junctions or none at all (McNutt and 1-leinstein, 1973; Martinez1
Palomo, 1971, 1970; McNutt, et al., 1971; Beneditti and Emmelot,
1968; Emmelot and Beneditti, 1967).
The fact that solid tumor cells have reduced numbers of junetions suggests a possible change in the plasma membrane which could
i.n turn be responsible for differences in the adhesion and
behavior of tumor cells.
11
soci.al 11
One could depict the tumor cell surface as
being modified in such a way that the components which mediate the
formation of a particular type of junction are missing, in which
case one would not expect junctional formation under any set of conditions unless these components were regenerated at the cell surface. i
I
I
---···-··· ...... ...J
6
r------------------···------------------------·-·-----------·-···-------·---------------~--- ------------------~
I
lon
the other hand, these components could be present within the cell
lbut are not able to interact because of any of several reasons, such
I
as greater repulsive forces between the cells, or cryptic burying of
the components involved.
1
I1 ditions
Ishorter
1
Obviously, if the proper environmental con-
are met, the time required for formation of junctions would be 1
in the latter case since the components are present and do not
l
lneed to.be regenerated.
I
I
11
In vitro'' agglutination appeared to be a useful approach to
I
Iprobe the phenomenon of junctional complex formation in tumor cells;
Iagglutination acting to bring cells in close contact in the presence
Iof an
exogenous component.
I· Ln
. t erac t.Lons
In this study, the problem of membrane
. a mouse ascL. t es t umor 1'Lne,
Ln
s arcoma
180 (S - 180) , was
examined over short and extended periods in culture using thin section
j and transmission electron microscopy (TEM) after agglutination by
I
Con A, an ascites "factor", and manganese ion.
I
I
I,-----
----·-··-··-- ·-·-··---------·------------ ......- ..·--· ·-· -------···----·-------·-·
~-~----~
i
Methods and Materials
I Maintenance and
~-
preparation of S-180.
.
The ascites form of S-180 was originally obtained by Dr. Steven
I
•
/I
Oppenheimer from Dr. Melvin Cohen of the Salk Institute and from Mr.
I
I
Samuel M. Porley of the National Cancer Institute.
~
I by
I
intr·aperitoneal transfer every 4-6 days in Swiss··Webster, young,
male mice.
I ascites
The abdominal skin \vas opened and the
fluid was aspirated with a syringe bearing a 19 gauge needle
1
I
I
1
I·
'~i;
Tumor bearing mice, inoculated 4-6 days prior, were kill-.
ed by cervical dislocation.
1
It was maintained
or by cutting open the peritoneum and pouring the ascites fluid dir-
ectly into a plastic petri dish.
I
!
i
j
The cells were harvested and washed
tHice by low speed centrifugation in Hanks' Balanced Salt Solution
,1
I
I'
(BSS) before use at an appropriate dilution.
I
l
Viability Test.
Viability was determined using the Nigrosin dye-exclusion test.
The test \vas performed by mixing a sample of cells with an equal
volume of a 1% solution of Nigrosin in Hanks' BSS.
The cells \vere
examined after five minutes under a light microscope using low power.
Il
I
I
Stained cells were considered dead.
The percentage of dead cells
was determined by counting at least 100 cells.
Electron Microscopy.
Fixation of cells was carried out in either a 1:1 dilution of
Karnovsky's fixative (Karnovsky, 1965) Hith 0.1 M sodium cacodylate
____ j
7
l
8
..·---·····-·
r·~
··--~
.................
---~--.--
.........------------·-·. ·-·----·-·------·--···-··-----· ....
......____1
·~---
il
I
I
I
I
I
buffer pH 7. 4 (Shea, 1971), (referred to as "modified Karnovsky 1 s 11 } ;
1
1
I
i
times I
or with 2.5% glutaraldehyde in 0.06 M sodium cacodylate buffer at pH
7. 2-7 .4.
Fixation \vas at room temperature for 2-4 hours and at
continued overnight at 4°C.
The cells were washed three times in
I
I
O.lM cacodylate buffer pH 7.4 then postfixed in 2% osmium tetroxide
in 0.1 M cacodylate buffer pH 7.4 for two hours.
The cells
~vere
then
washed twice in cacodylat·e buffer and dehydrated in a graded series
of acetone.
Blocks were sectioned on a Porter-Blum MT2 Sor-
vall ultramicrotome and stained with 2'7o uranyl acetate in 50% acetone
and Reynold's lead citrate (Reynolds, 1963).
Sections
~vere examined
!
I
I
added to enhance staining of the glycocalyx.
!
I
1
I
Lanthanium Oxide Staining.
Only the
Cells
were fixed in modified Karnovsky's fixative with 0.1% Alcian blue
I
II
!
Con A-treated cells were examined using this staining method.
I
!
with a Zeiss EM 9S-2 electron microscope.
Lanthanium staining was as described by Shea (1971).
I
Il
The cells were embedded in an Epon-Araldite mixt:ure
(Mollenhauer, 1964).
i
l
Fixation was carried
I
I
l
l
I
out at room temperature for two hours.
Postfixation was carried out in 1% osmium tetroxide and 1% lanthanium nitrate in 0.1 M cacodylate buffer.
The final pH of the fix-
ative was adjusted by gradually adding sodium hydroxide (0.1 M) to
the solution while on a magnetic stirrer.
Sodium hydroxide ·was added
just until a small amount of precipitate was formed (pH about 7.4).
Fixation was carried out for t\vO hours at room temperature.
I
1
iI
!
l'
Dehydra- i
. . . . . ···-------.. . . . -.. . . . . . . . . . . . . . . . ._. . . . . . . . . . . . . . . . . . . . . . ----··· . . . -.. . . . . . . . . . . . -.. . . . . . . . . . . . . . . . . . . __~. . . . . . . . . . . ..J
9
tion was carried out in a graded acetone series, 1% lanthanium nitrate being added to the lower dilutions in the series.
Embedding
was as before.
Ruthenium Red Staining.
Ruthenium Red (RR) staining was performed as by Luft (1971).
Only the manganese-treated cells and untreated cells \vere stained in
order to enhance their glycocalyx.
A stock solution of RR was made by adding RR to distilled water
(lOmg/ml) and heating to a boil.
This solution was cooled, placed in
a centrifuge tube and spun in a clinical centrifuge for ten minutes
at full speed.
Supernatant was removed and used as a stock solution.
Staining was accomplished in the fixative by using a 1% solution
of RR stock in 2.5% glutaraldehyde in 0.06 M sodium cacodylate buffer
pH 7.3
The cells were fixed and stained for 4 hours at room temper-
ature, then washed in 0.1 M cacodylate buffer followed by postfixation in 2% osmium tetroxide for 2 hours.
bedding was as before.
Dehydration and em-
Sections were examined without any further
staining.
Concanavalin A Agglutination.
Con A agglutination was performed according to the method of
Oppenheimer and Odencrantz (1972).
Equal numbers of washed cells
were agglutinated with either a 500 or 1000 pg/ml solution of Con A
in Hanks' BSS.
The cells were suspended in 2 mls of the Con A solu-
tion or in Hanks' BSS, in screw-capped scintillation vials.
The
I
·- --· ·-- - - ··- -~··---...J
10
i--·········-··-------~-----·-···---·····-·------···----------·--------------···-·--------·····--------····----,
!
!vials were immediately placed on a rotary shaker having a 4-5/8" dia-
l
!meter of gyration and rotated at 65 rpm in a 37°C incubator for 15 -
li60
minutes.
At the end of this period, the cells were washed with
several milliliters of Hanks' BSS and collected by slow centrifugaltion.
The pellets were resuspended in a 1:1 mixture of Hanks' BSS
!containing 5 pg/ml of deoxyribonuclease (DNase), and Eagles Minimal
!Essential Media (MEM) without glutamine and buffered with 0.01 M
IHepes, pH 7.4
The resuspended cells were then incubated in 25 mm
!plastic tissue culture dishes for one and four hours at 37°C.
The
!
!cells were collected and fixed for electron microscopy with modified
II
!Karnovsky's fixative.
I
i
iFactor Agglutination.
I
Ascites fluid from tumor bearing mice was collected as before.
I
i
!Cells were removed by centrifugation and the supernate was used as a
I
crude "factor" preparation.
I
!
_Ill
Hashed cells were agglutinated in a 10% or 37% mixture of the
crude factor preparation diluted with Hanks 1 BSS with DNase (5pg/ml)
I
added and using a rotary shaker as with the Con A agglutination (Oppen-!
jheimer and Humphreys, 1971).
After 15-60 minutes, the cells were
I
!washed with Hanks' BSS with DNase and pelleted by centrifugation or
by settling.
I
The pellets were resuspended in a 2:3 mixture of Hanks'
BSS with DNase, and Eagles
~~M
without glutamine, incubated in 25 mm
I
plastic tissue culture dishes for one or four r.ours, and fixed with
I
, modified Karnovsky's.
'
L .. · - -··-----·--·-··--····-·· ··-- . ···---· ----·····-----·--··----·-· ··-···---~--·-- - ·-- ·- -....... _. ----··· .. -·-----···-······
... -- -- .. J
I
ll
r·--·----------·-----·--·-··---·--------------·-··.....------·---------·---·---..
~---·---------·--------·----------- -----~1
I
I
I
I
----................._.......J
r------ -----·-----· -----·-·---------------------------------·--·--------- --·----------------------------------·. ·----------------..,
I
!
Results
i
I
I
Fine structure of S-180.
The diameter of S-180 cells is generally
15-20~
but a variable
percentage (about 5 - 15%) of the population is found to be
25-3~
1
1
(Plates lA & B).
Plate 2 shows S-180's general appearance.
Structurally the most
prominent features are a large, highly fissured, multi lobate nucleus
I
(Plates lA & 2) and a very active cell surface with numerous villus
·
projections (Plate 2).
I
The nucleus characteristically has many nucleoli.
Most are as-
l
sociated with the nuclear membrane at the distal ends of intranuclear!
I
inuclear and closely associated with the nuclear membrane. Many fis- I
I
sures or deep invaginations of cytoplasm course through the nucleus
caniculi (Plates 3A & B).
(Plates 2 & 3).
inclusions.
Heterochromatic regions are normally per-
In addition, one occasionally sees several nuclear
In Plate 3 for instance, one sees clusters of granules,
!
I
1
possibly viral cores, and what appears to be membrane bound vesicles. I
The nucleoli are large.
They distinctively have a large, light-
I
staining pars amorpha, and extensive, dark-staining, granular pars
granulosa.
The pars fibrosa is small but characteristically is
I
found between the.pars granulosa and amorpha (Plate 3A) (Goessens
and Lepoint, 1974).
A region of chromatin (with caniculi) can be
seen associated with the pars granulosa (Plate 3A).
The nuclear envelop is similar to that of normal cells.
The
... ---·-··- ····----- -- ·········----------··· . -----.1
12
13
,--··"----..---·--·--··· .......,... ,- ··-···--- ___,_..... -· ..... ·---------- "' ............----·-·---------------'"""-'""""'"''""··1
'
l
double membrane being typically interrupted by nuclear pores (Plate
3A).
The cytoplasm tends to be dense with large numbers of free ribo-
II
somes characteristic of tumor cells, and large numbers of mitochondria.
The mitochondria are quite pleomorphic.
They are most common-
1
ly tubular with a moderately dense and granular matrix, with typical
cristae,- though they may be club shaped or S\.;rollen in appearance
(Plate 4).
Less commonly, the mitochondria may have a vesiculated
matrix having a lighter appearance but w·ith typical cristae (Plate 2),
and occasionally mitochondria may have a dense granular matrix and
vesiculated cristae (Plates SD & 7B).
due to the energy state of the cell.
types reflecting the
11
orthodo~'
This pleomorphism is likely
The first and maybe the second
or resting mitochondrion and the
third type, with dense matrix and vesiculated cristae, reflecting the
"condensed" or rapidly respiring mitochondrion.
Rehn, 1970).
(Hackenbrock and
The mitochondria are generally distributed uniformly
throughout the cytoplasm though it is not uncommon for them to be
found somewhat more peripherally.
The centriole and Golgi complexes are found in the centrosphere
(Plate 4).
One to three Golgi complexes are commonly seen in a sec-
tion, always associated with a large number of vesicles and vacuoles
suggesting activity (Plate SA).
Often seen in the region of the
Golgi are various darkly stained bodies (Plate SA).
The amount of endoplasmic reticulum (ER) is somewhat varible,
I
l
but usually is not extensive in these cells.
1..~-"·-~··'"
I
I
Some smooth ER is seen, 1
--·---·--• ~--·-.-·- -·•- --~···--·• -··~------~···-- ••••·-- .. . .rl
14
rough ERpredominates.
The ER is usually filled with a moderately
j staining material (Plate SA).
I
Annulate lamellae are seen but rarely.
Various types of lysosomal vacuoles are seen in these cells.
!Plate SB shows what appears to be the fusion of residual bodies with
!
Ia digestive vacuole.
Autophagic vacuoles can likewise be seen
I
ii (Plate
12D) .
In the presence of high concentrations of exogenous
Iproteins -(in this case ascitic fluid)
Iseen
(Plates 12B & C).
numerous endocytic vesicles are
These probably fuse with numerous lysosomal
i
I vesicles
II can
in this region, forming the numerous residual bodies which
be recognized by the halo between the contents of the residual
I
I
body and the delimiting membrane (Novikoff, 1973).
I t was not un-
common to see large myelinoid-type residual bodies in these cells.
1
II These myelinoid
figures appear to be readily exocytosed by the cells
I
(Plate 12D).
i
I
I
Many large, spherical, lightly-stained bodies can be seen in
these cells.
They do not appear to have a delimiting membrane, and
i
appear to be readily exocytosed (Plates 5C & D).
II
lipid droplets as they appear similar to those described in other
These are probably
tumor cells (Chambers and Weiser, 1964; Bergstrand and Ringertz, 1960;
I
! Epstein,
~
~·
1
II
1957).
I
In addition, there were other large spherical bodies with greater j
electron density.
Some stained irregularly (Plate SE) and others
1,.
appeared to have a halo of densely stained particles surrounding them '
(Plate SF).
figures.
!'
The latter type was frequently associated with myelin-likel
No delimiting membrane could be seen around these bodies,
15
~-------.
--- -----------------·------ -·--
-- ----·
---- -----·--- ---
---
----- --·---------------
·---------------------,
I
I
Iibut
I
the membrane may have been obscured by the density of the stain.
lit was not uncommon to see the first type in clusters of two and
I
~three.
Apparently they coalesce together in the cytoplasm.
Rarely, one sees aggregates which appear to be viral capsids in
j
!
l the cytoplasm (Plate 6A), and individual capsids in the ER.
j
The cap-
I
Isids are about 50 m~
II size and ·symmetry
i
I et al., 1973).
in diameter with isosahedral symmetry.
Their
suggests they belong to the Papovavirus group (Davis,
II
i
The cell surface is quite variable.
Nost commonly the surface is
i
i
! covered
I
'
i
I
with numerous microvilli of varying size, shape and number.
I
l
These may appear thin and finger-like or thick and club shaped (Plate
I
i
I 6B).
The distribution of microvilli on the cell is not always uniformJ
!
i Nembrane loops are common, and endocytosis can be seen.
I
I
Concanavalin A agglutination.
!
After removal from shaker treatment with 500 or 1000
Con A, cell aggregates were seen.
~g/ml
of
I
These ranged from a few millimeters!
!
across, down to two and three cell clumps in addition to unagglutinated, single cells (Plates lC & D).
Hithin approximately four hours,
the large clumps had broken down into smaller aggregates of several
cells.
Longer incubation (20 hours) resulted in low viability of the
cells.
The decrease in aggregate size is probably not affected by
the dead or dying cells, .as they \vere tightly associated with viable
cells (Plate 6C).
The agglutinated cells
~;.;rere
examined ultrastructurally at one,
J
i
16
,-------- ------------·----·--··---------------------------------------------·-·-----·---· ' -------· --·-·····---------" __. . ---------------1
II four
I
and t\venty hours after treatment.
In general, the viability of
the one and four material, as determined by dye exclusion, was high,
j but
I~
I
consistently too low in the twenty hour material to be meaningful.
This loss in viability could have been due to the cytot6xicity of
Con A, especially since it was used at possibly toxic levels in order ·
I
to maximize agglutination for electron microscopy.
Also
growth con- )
ditions may not have been optimal, as later work suggest: these cells
require a rich medium initially after transfer to
11
in vitro" cultures.!
Three types of cell-cell interactions were seen between the Con A
agglutinated cells.
I
I!
The interactions after four hours \vere similar to
those seen after one hour.
The interaction could be classified into:
1) Those mediated by microvilli-microvilli and microvilli-cryptic
l
surface interactions; 2) intermediate associations usually of the
focal type, in which the membranes are separated by about 100-250 A
and between which a lightly stained material is usually seen; and 3)
close associations in :hich the membrane of adjacent cells are separ-
~;:
ated by less than 100 A.
_
Plate 7A demonstrates the first type of interaction and compares
Con A treated and untreated cell surfaces (Plate 7B).
treated cells seem to have few projecting villi.
The Con A
Many villi which
can be seen in cross sect:ion apparently have been agglutinated by the
Con A to the parent cell surface and to the villi and cryptic surface
of the adjacent cells (Plate 7A).
Membrane loops can be seen on both
Con A and untreated cells, but are accented on the Con A cells by the
lack of projecting villi.
The obvious projection of microvilli on the
l
l
I
. ·-----------.. -----...- -................ ____ -- ................. ,...... J
17
,---------- --------·· ·-------··---·-·------------.--------·-----·-------- ... _________,__ -·- ------·------------.. . . .l
I
I
~-.
!control cells with few villi-villi or villi-cryptic surface inter!actions is quite apparent; in most instances no associations are seen
I
~between
i
I
I
untreated cells (Plates lA & B, 2).
How stable the microvilli mediated attachments are is unknown,
!but similar associations are seen in cells cultured four hours {Plate
j8A).
Plate 8B shows how extensive these associations can be, some
I
I v 1."11"1.
actually seem to be pushing_into the adjacent cell producing a
very tight fit between surfaces.
This situation 'vas not seen very of-
ten, and only in the four hour preparations, though it may have been
present at other times and not observed.
The villi normally appear
I
~.!
i
I
to I
'
be lying flat, apparently agglutinated to the surface of one or both
I
adjacent cells, and many times lying between cells that are possibly
!
being held together by areas of closer association {Plates SA & C).
The intermediate associations are very common and characterized
by the adjacent membranes being separated by greater than 100 ~-
I
i
They I
usually have a lightly stained material in the intercellular space,
and do not appear to be extensive in area (a focal type association)
(Plate 9A).
The adjacent membranes are approximately parallel.
This
type of association is most commonly seen in areas which hold the
cells together in clumps (Plate 8A).
Plates 9B and C shmv that multi-
ple points of interaction can occur between adjacent cells.
The com-
mon situation consists of large regions in which focal intermediate
associations appear to be holding the cells together.
I
I
I
'
In the inter-
vals bet,veen these associations, the membrane appears irregular, commanly with villi projecting into the open spaces.
l
i
The cell surface is 1
______ ..., ............. -- ---------------·" ......... -· .......... .J
18
r-------·---------····-·--
···--···--·-·--~---------y
Ii
Ifrequently characterized by amorphous
debris possibly derived from
I
I
I
!the media.
!
There is no type of cytoplasmic specialization found in the intermediate type of association comparable to the classical zonula adherens type of junctional complex.
But these may represent a modifi-
!cation of this junction, and it may form as a result of the cells be-
l ing
brought into close contact by the lectin during agglutination.
!
I It
is not knmvn if Con A is found between the cells in areas of these
I
Iassociations.
I
The last type of association seen, the close association (Plates
I10 A-D),
I separated
J
is characterized by the membranes of adjacent cells being
o
by less than 100 A.
Imembranes can be estimated
II thickness
I
1 lOA
is about 70
R.
In Plate lOA, the gap between adjacent
to be about 20
Xassuming
the membrane
These may appear as either focal (Plates
& B) or zonula (Plates lOG & D) types of interactions.
No cyto-
i
!plasmic specialization is seen, and the outer leaflets of the opposing
~membranes
I' ly
1
i'
do not fuse at any point.
common; especially the focal type.
teristi~ of
the gap junction.
This type of association is fairIts appearance is very charac-
Attempts to use the characteristic lan-
lthanium oxide staining property of gap junctions failed to answer this
1 question.
ilremaLn
. Ln.
!
1 In
It was felt this resulted from the failure of the stain to
the loose cell aggregates during washing and dehydration.
no case were tight junctions observed in \vhich the outer leaflets
1
lof the adjacent membranes would have fused, nor were desmosomes with
!i their
characteristic cytoplasmic tonofilaments seen in any of these
L____ ---- ---··-· - -·------------- ---· ·- · · . . · ---- - - - - - - - - · - - - --·-- -·------ -· - - -· ---------.. - - - - - - - - - - - - - - - _________j
19
.
r···~----...-..-- ---~----··· --~" --------~-------------
- -----
~-·------
---- ---------·---
- -------·-· -·- ··--·- -- --------·-- --- -------,
materials.
I
Factor agglutination.
Two dilutions of "factor" (Oppenheimer and Humphreys, 1971) were
used:
10% and 37% in Eagles MEM.
the concentration used.
The results were dependent upon
At the higher concentration after one hour
(Plate 11) the cells actively phagocytosed a large amount of flocculent material from the medium.
This material also appeared to be ad-
hering to the cell surface (Plate 12A).
Using high factor concentrations, the cells aggregated to form
large clumps, but there appeared to be no type of close cell associa-
I
I
I
I
tions.
Moreover, it appeared that agglutination in this case
~vas
l
I
II
i
i
due to large amounts of material attached to the cell surface,
which crosslinked the cells together (Plates 12B & C).
Hmvever, it
should be noted that the factor used in these experiments was relatively inactive, requiring high concentrations for cell binding
activity.
The cells appear very vesiculated as a result of pinocytosis.
Many lysosomal vesicles were obvious and the rough ER had become
branched and slightly swollen with a moderately stained material
(Plates 12B & C).
The number of villi had decreased, possibly in
response to the large amount of membrane involved in pinocytosis.
At the lmver concentration of "factor" a much different situation was seen (Plate 13).
The cells appeared normal with large
numbers of villi and reduced amounts of roughER.
In this case intermediate type associations similar to those
I
20
r~--··--------
. --·-··------------------........ · -· --------..·--------·-·----·-----.. --·-·----..--------------- ·-·-------- . ----------l
Iobserved with Con A were seen between the cells (Plates l4A-C). A
I lightly stained material was obvious in some patchy areas (Plate l4D),'.
,I
I
and in many cases villi were absent from the intercellular spaces. No
gap or tight junctions were apparent in these preparations.
The material in these associations could be the component(s) of
the ascites which \vas responsible for agglutination and may be crosslinking
t~e
cells together.
Shepard and Oppenheimer (unpublished ob-
servation) have shown some degree of specifity of the "factor" for·
these cells which may involve terminal galactose resides on the factor!
binding to cell surface receptor sites (Oppenheimer, 1975; Oppen-
'
heimer, personal communication).
With time in culture, the cell aggregates tend to breakdmvn.
Af-
i
ter four hours the number of associations decreased. ·Most cells appeared singly and looked like untreated cells with numerous villi and
lobated surfaces.
It could be that the components responsible for
agglutination are removed from the medium and cell surface with time,
possibly by pinocytosis and phagocytosis.
Factor may have to be re-
plenished to maintain the effect.
I
I
Plate 12D shows two cells, one of which is apparently exocytosing!
a large myelin figure.
The complementarity of the cells' surfaces
suggests they are being forced apart by the myelin figure.
I
I
This
could suggest that the strength of the association is not very great
; and can be broken by any projection from the cell surface.
Also,
since the cells have remained nearly complementary, cytoplasmic stabilization of surface shape may be an important aspect of adhesion.
L._.............-----·- ------------·- -------------- . --- ---------
________. ___ ------------------------ ----------~------------------ --------- - - - . .... ____ _}
21
~---··«•·······--·
·-··-----·· ----------·· --·······---·---·--·-·········------···-- --------------- .... ----·------------- .... ---------·
---·-------~
,
II
jManganese ion agglutination.
Manganese ion produced a very rapid agglutination of S-180 into
large clumps.
I! immediate
I utes.
The initial events in agglutination were examined by
fixation followed by additional fixation at 3 and 30 min-
Light micrographs show clumping of the cells after fixation
1
Iand infiltration with
I
\
I
plastic (Plates 15 A-C).
Ultr~structurally
Ibetween the
i minute
no differences were seen in the association
initial (Plates 16 A & B), and 3 (Plates 16 C & D) and 30
(Plate 17A) preparations.
There did not appear to be any dif-
1
ferences in the Ruthenium red staining properties of areas of inter-
i
action and noninteraction (Plate 17B).
1,11_
Plates 17 C & Dare high mag-
1
,
j nification pictures of the interactions occurring between adjacent
i
! cell
surfaces. The distance separating the membranes is very small,
I
a
! less than 60 A and in many cases no distinct separation can be seen.
I
\ While this suggests some type of primary interaction between the mem-
l brane surfaces,
I
ionic bridging between components on the surface of
the villi seems likely.
I change is
I
cells.
These interactions appear static since no
seen in them between the initial, 3 minute, and 30 minute
The strength of this interaction is not known, but probably
requires the continuing presence of Nn
-1+
.
After four hours in 20 mM Mn-1+ in serum containing medium, the
cell aggregates were dissociated (Plate lSD).
I
Most cells seem swollen!
}
(Plate l8A) and large numbers of vacuoles and invaginations are appar- 1
.
ent.
i
Plate 19A shows how deep the invaginations of the cells can be.
The surfaces of the cells were quite variable.
'----- ... ··-- ..... -- ---·-··· --------------. -
-······ ----------
The cells appeared
I
!
l
i
.......... ----- .............. -- ······.- -- .................. ------ ...........!
2.2
IIcompletely smooth
/villi (Plate 18C).
(Plate 18B), lobated (not shown), or with numerous
The cytoplasm is denser around the nucleus in
!
many cells and the mitochondria are condensed, deeply stained, and
with s1;.mllen cristae.
The breakdown of the aggregates may, in part, be due to the loss
of villi from the cell surface, but the morphology of these cells is
quite unusual and may reflect a pathological situation as a result of
the relatively high level of manganese ion in the medium.
The associations that occur with Mn++, while similar in some
II ways
to those seen after Con A agglutination (Plate 7A),
I
i
I
to lead to flattening of the cells' surfaces nor to more extensive
I interactions
Ia
between cells.
situation unsatisfactory for further involvement by the cell sur-
1 face.
Ii
The relative staining intensity of the microfilaments within the
I
microvilli is greater after Mn++ treatment (Plate 17D).
1 gests
This sug-
Mn++ may be interacting with microfilament components.
I
It is difficult to quantify the occurrence of each type of association in this study.
I
I
l
However, using micrographs of situations
which were judged to be typical, Table 1 was constructed to indicate
I
the relative occurrence of each general type of association after
treatment with each agglutinin.
I
Use of the terms 'close', 'inter-
mediate', and 'microvillous' does not imply that the agglutinins act
in similar Hays or that the fine structure of each is exactly identical.
t
~~--~---~-------
-···· ·-
....,~--····
-·-------···-------~
-----. ·-··---· ··--·--··· ···--~ -------·-·- ------- -----·-------··
~
-----
---
I
I
I
__________ __~
23
i ------- -·
---~·------------
--------·-·------------ ----------------------------------------------------- - ------------------ "---
-------1
I
I
I
Each association may occur alone or in combination with the
II
I ::::: :::e:~di:::: ::g:::·o::::•:y::.h::ea:::::::::::·.:·:::i::::::e I
I
I
in Plate 14A where two microvilli appear to be associated with the
adjacent cell by an intermediate association.
In the case of mangan-
!
ese) villi interact by associations which appear closer than the
close associations seen with Con A.
l
i
!
l
I
........ ------.. --- . . ---------------·---·- .... -___ ...l
r------·-··. ------------------ - ·- ------- -· -------·----------· --- ---------· ------ -.---------- -------------..·--------------------·-------··:
l
I
I
Discussion
'
The fine structure of S-180 has been reported previously by
Molnar and Bekesi (1972).
This work expands on their observations
and reports some differences.
S-180 shows the general structural characteristics of most tumor,
cells'
The large, irregular 1 y shaped, heterochromatic nucleus; the
I
large number of surface villi; the large number of free ribosomes,
I
and decreased amounts of ER (Molnar and Bekesi, 1972; Chambers and
Weiser, 1964; Oberling and Bernhard, 1961; Bergstrand and Ring·ertz,
1960; Bernhard, 1958; Epstein, 1957; Dalton and Felix, 1956).
In addition, it shares some structural characteristics of other
I
!
ascites tumors.
!I
Large lipid droplets \vere described in Sarcoma 1,
Sarcoma 37 and MClM tumors (Chambers and Weiser, 1964; Bergstrand
and Ringertz, 1960; Epstein, 1957.
by membrane.
These do not appear to be bound
They tend to show a light, uniform staining, and may
also show a granular darkly stained periphery.
Similar lipid bodies were seen in S-180 (Plates 5C & D).
As
seen in Plate 5D these lipid bodies could be readily exocytosed.
In
addition to the bodies described above, there \vere other large, darkly stained bodies (Plates 5E & F).
These did not appear to be mem-
brane bound, but the membrane could have been obscured by the density
of the stain.
Whether these represent pleomorphic forms of the form-
er type of lipid body, or a different type of vacuole was not determined.
The large size and apparent lack of a delimiting membrane
24
25
suggest they are lipid bodies.
I type of residual body.
I
I type
The association of myelin figures \vith one
(Plate SF) may be suggestive of this, but residual bodies are
!bound by a tripartate membrane (Novikoff, 1973).
A cytochemical an-
;alysis will be needed to characterize these bodies.
I!
Viral capsids occasionally occurred in the cytoplasm of these
cells.
Eowever, virus particles were not seen budding off the cell
membrane and this suggests they are not an enveloped virus.
Their
size, symmetry and probable lack of envelop suggests they belong to
the papovavirus group.
Members of this group are commonly seen in a
small percentage of cultured mouse cells and some tumor cell populations (Davis, et al., 1973; Sanders, 1973) and similar virus particles have been seen previously in S-180 (Molnar and Bekesi, 1972).
C-type viruses were not seen.
The large number of microvilli on the cell surface is a common
feature to ascites and also some solid tumors (Chambers and Weiser,
1964; Mercer and Easty, 1961; Bergstrand and Ringertz, 1960; Wessel
and Bernhard, 1957).
number and shape.
S-180 shmvs numerous villi of various lengths,
They could be irregularly, or uniformly distrib-
uted on the cell surface.
While these appeared to be important in
the cell-cell interactions described, regions of extensive interaction were not seen between untreated cells.
The S-180 cells used in this study characteristically have few
microfilaments.
On the other hand, bundles of fine filaments have
been described in Sarcoma l, Sarcoma 37, :tviClM; and also in fibro-
I
l
......,. ---------- .......... J
26
r--·---------------- ------· . ----· -------- ·---- --------- ·---- ... ---------- --------· .. --------------------------· -------------- -- . -- ---· ------ ---------1
i
blasts and as the tonofilaments of epithelial cells (Molnar and Bekesi, 1972; Chambers and Weiser, 1964; Journey and Amos, 1962; Ross
and Benditt, 1961; Bergstrand and Ringertz, 1960; Epstein, 1957;
Selby, 1955).
The lack of cytoplasmic filaments in this study is in
contrast to that of Molnar and Bekesi who observed them in the S-180
they used.
This difference may relate to the particular line of S-
The ultrastructural features of the agglutinated S-180 cells
differ depending on the agglutinin and this in turn on the action of
the agglutinin on the cell surface.
In the Con A system, the cell-cell interactions were of three
types:
1) Microvilli-mediated, 2) intermediate, and 3) close asso-
ciations.
These were seen after both one and four hours.
It should '
be noted that aggregates, formed initially after treatment with Con
A, were relatively large when examined by eye and these dissociated
into smaller aggregates with further incubation.
This would suggest
that many of the interactions are \veak, reversible, or subject to
degradation possibly of the Con A or its receptor.
In addition,
further dissociations occurred as a result of the preparative steps
for electron microscopy.
Glutaraldehyde fixation did not stabilize
the interactions between the cells.
The smaller aggregates may rep-
resent only the stronger or more extensive associations that form
and which are able to survive the technique.
Several workers have shown Con A receptor mobility (clustering)
to be related to agglutination (Rutishauser and Sachs, 1974; Rosen........ --···--·· -'- ---------------- __________j
7!.7
r--·---···----~----··-------~~---
-- -------- -·- -· --· - ----·--·-··--------· ----------·------.... ----·-· - . ------------··--·. -- ··-·-l
i
blith, et al., 1973; Nicolson, 1972; Nicolson, 1971).
The types of
associations seen here may represent clustering of Con A receptors
Ii
resulting in crosslinking of the cells, or some type of Con A initi- I•
ated interaction between the cells.
Host of the work on Con A recep-
II
i
tor mobility and clustering involved examination of free surfaces of
cells and not the interaction between cells.
There does not appear
son (1972) has examined, by thin section, the agglutination of 3T3
cells after proteolysis, while paying heed to mobility of receptors
Proteolysis stimulates agglu-
tination between normal cells which agglutinate poorly at high lectin
concentration.
His work shows a greater concentration of ferritin
conjugated Con A between the protease-treated 3T3 cells at the
points of contact than on the free surfaces.
Moreover, the ferritin
Con A appeared in the region of contact to be clustered into patches
crosslinking the cells.
The agglutinated cell surfaces were separ-
o
ated by 200 - 400 A.
Cell aggregation could be reversed in this case
by a-methyl-mannoside when added shortly after agglutination.
Lee
and Feldman (1964) reported a similar gap between human red cells
agglutinated by ferritin conjugated group A antibody.
The first interaction, the microvilli-mediated association,
could represent initial contacts betHeen cells \vhich then develop into the stronger types of associations.
and, therefore, can dissociate.
IL._.that
I
Nicol~
to be any work examining the interactions between tumor cells.
on the free surface of these cells.
I
'
They may also be reversible
There has been some work showing
the microvillus surface is chemically different from the inter-
;
28
r~-----------
--------------.
I'
villal membrane (\veiss and Subjeck, 1974).
These differences may be
reflected here but they are difficult to assess.
The suggestion of
villus interactions leading to the more extensive types of associations is difficult to reconcile with what is observed here.
The
villus interactions are seen in both the one and four hour material.
It would be expected that many of these would have progressed into
the other types of associations by four hours.
Intermediate steps
leading from villus interaction to the extensive close associations
are not obvious in this material.
The villi interact with cryptic
cell surfaces and other villus tips by both intermediate and close
associations.
This may indicate that the interactions of the villous!
and cryptic cell surfaces are comparable to the interactions that
occur in regions where membrane flattening has occurred.
There did
I
I
I
not appear to be any cytoplasmic or intercellular specializations oc-!
I
curring at the site of interaction.
An increased staining density
was observed at the site of microvilli interaction in Sarcoma 1, "in
'I
I
vivo" (Chamber and Weiser, 1964). One would expect the villi to inter-1
act extensively after treatment because of the large number of villi
II
on S-180, and also because they would be involved in the initial con-
I
tact s be tween the cells.
It is d iff icu 1 t to discern whether this
I
first type of association is independent in the agglutination process l
or plays a transistory role in the formation of the other types of
I'
i
Hssociations.
I
I
The intermediate associations seen in this work resemble the in- !
I
teractions described by Nicolson between 3T3 cells.
t~---·~·-
The associations j
I
·-···
.. ---·---·~-- ~-
.. ------··-----·- ···-··--··- .............!
29"
r···---···- ------· "--- . ..
~--
'_1
~·--
'----------·---··--·-¥--~~------·
--- ---------------~-
--··-~----·---~---··----------·~-------.
.
---~--
..
--------~------~~--
.l
in this study are characterized by patches of a lightly stained, mem-
1
l
! brane
associated material crosslinking the S-180 cells.
I very \vell be aggregates
This may
of Con A (Agrmval and Goldstein, 1968) agglu-
tinating the cells together.
The patchy nature of the interaction
does suggest clustering of Con A receptors as a requirement for agglutination in this situation as in the 3T3 study.
Another interpretation of the intermediate association is that
it represents some type of junctional complex which develops secondarily as a result of primary Con A agglutination.
While these asso-
ciations do appear similar in some \vays to the zonula adherens junetions of normal cells, no cytoplasmic specialization is seen in S-180.!
The junctions would normally have actin-like fibers associated in a
j mat \vithin the cortical cytoplasm under the plasma membrane.
I
The ab-
1 sence of fibers and the general patchiness of juxtapositioning does
I
suggest this association is not a zonula adherens, but represents the
1
crosslinking of the cells by Con A.
I!
ruled out by this work (McNutt and Heinstein, 1973; Pinto-daSilva and
Atypical junctions cannot be
j Gilula, 1972).
l
'
Close associations were not reported by Nicolson between agglutinated 3T3 cells.
As seen in this study, these may represent regions.,.
of Con A crosslinking dissimilar to those described by Nicolson, but
it is not likely that the close associations, especially the extensive ones, (Plate lOC), would represent areas held by Con A.
is a polymer in solution.
Con A
The size of the basic protomer as detero
mined by Edelman et al., (1972) is 42X40X39 A. Above pH 7, Con A is
I
30
r·---·--·······------- --------·---·----········-··- ........ -------- ---- ... ----······ ...... ---------·--------·- ------- -------·----------- ----~
I
found in the form of tetrameric and larger polymeric aggregates of
I
the protomer (McKenzie, et al., 1972; Agra\val and Goldstein, 1968;
Kalb and Lustig, 1968).
0
20 A.
The gap in the close association is about
Thus, the dimensions of the aggregated Con A agglutinating the
cells (Con A tetramers and larger polymers) would be greater than
that of the gap.
This would argue against the direct involvement of
Con A in the close association.
Rutishauser and Sachs (1974) reported some work using Con A
; derivatived nylon fibers which shows the cell-cell interactions between two lymphoma tumor cells after Con A agglutination is not reversible by a-methyl-mannoside.
It was suggested by the authors
that Con A acts to bring the cells into close contact at which time
cellular components can interact to produce a mannoside-insensitive
binding.
These interactions were not examined ultrastructurally by
the authors.
The work here is suggestive of a comparable situation,
though reversibility was not examined in this study.
The extensive
regions of close contact could represent regions in which strong
cellular interactions have occurred; these associations being induced
in some way by agglutination, but \•lith cell surface components inter··
acting.
The appearance and dimensions of the close association very near-·
ly resembles the gap junction or nexus.
An attempt to use lanthanium
oxide staining of this association failed to produce any staining. It
is possible the aggregates were too loosely associated to allow· the
stain to remain betHeen the cells (Revel and Karnovsky, 1967).
iL_ ____ ..
This
31
,-~---------···-----·
I
I
!
--··--··--·------·--·-·-·-------------- ···-
--·----··--·-···---·-- --·-· -·· ·-··- --·--·--···-·······-···.
--- ·-------------------,
1
~-
question could be approached by freeze-cleavage techniques which
,I
should demonstrate the characteristic hexagonal array of membrane
l
1
I
components in the gap junction.
Whatever the nature of the component(s) involved in the close
I
association, they are present or inserted very quickly after treatment with Con A.
f
This can be implied by the fact that close associ-
ations ·are seen after just one hour.
If these components were being
synthesized "de novo" one would expect the close association to develop later, possibly after four hours.
interact
11
in vivo" is not known.
Hhy these components do not
Con A may allmv the uncovering of
cryptic sites, or the overcoming of physical restrictions.
A relationship has been demonstrated between lectin binding and
cell behavior.
Burger and Noonan (1970) used Con A to inhibit trans-
formed cell growth, and Friberg and his group (1971) \vere able to
shmv inhibition of migration.
This type of "social" behavior is
thought to be a form of cell-cell communication mediated by gap junetions (Bennett, 1973; Lowenstein, 1973).
It might be speculated that
what \vas seen by these groups represents a Con A induced coupling of
cells through the gap junction-like close associations seen here,
I
allowing for cell-cell communication.
Agglutination by the crude ascites "factor" at high concentratio~
l
resulted in large amounts of amorphous material adhering to the cell
surface resulting in clumping.
The interaction of this material with
the cell surface is probably nonspecific, binding in many areas of
the membrane inducing pinocytosis of the material.
The flocculent
•••-•-•
• - - - - · - • • " c"-'••
I
-~-J
32
--- ···---····--··· ... .
. .•..•..... ··-·-- ······- --······ .... ·•·····
.... ·-·-···-·
··------·-·.. - ....
.
-·····
-·-· ........ ·------ ... - .....
.,
-- --·······--.
·····----------··
.,
l
l
material probably includes, in addition to "factor", other ascites
proteins such as fibrin.
The method of obtaining "factor" may in
fact activate some of the clotting proteins resulting in their deposition on the cell surface.
l
I
One would not expect to see large
amounts of proteins adhering to the cell surface "in vivo".
In fact, 1
this material is not seen on cells fixed in ascites fluid immediately
I
after removal from the peritoneum.
I
At lower concentration, the large amounts of flocculent material
I
were not present and a situation develops similar to the intermediate
association seen with Con A.
These interactions appear to be medi-
ated by small areas of cell surface in \vhich a lightly stained material is obvious and, presumably, is the "factor" from the ascites.
The patchy nature of these interactions and the lack of surface interactions
in the regions without this material could suggest the
involvement of membrane components or receptors.
These may be asso-
ciated as patches in a mosaic on the cell surface.
Otherwise, the
factor may promote the clustering of surface receptors in a manner
similar to Con A and other lectins.
The factor isolated from a
teratoma cell line by Oppenheimer and Humphreys (1971) appears to be
a glycoprotein, possibly synthesized by the mouse, the tumor cell,
or both, and appears to possess terminal galactose residues which
bind to the cell surface (Oppenheimer, 1975).
Prefixing cells with
4% formaldehyde failed to inhibit agglutination suggesting that with
teratoma "factor", active movement of membrane components is not
I
necessary for agglutination to occur (Oppenheimer and Humphreys,l97l)J
. ---. · · · · -· · · . . . --· ........ ···-·-----····--··--J
33
··---------·····-··-··-·-·-····-·-··-··~-----···-· • • « • ·---~
I
The S-180 ascites factor used in this study appears to possess
different charge and size properties from the factor isolated from
teratoma.
There also appears to be a difference between the single
cell form and the embryoid body form of the teratoma
11
l
Whether the
teratoma and S-180 "factors" are mechanistically similar is not
knm-rn.
I
Ij
factor 11 (Oppen- i
heimer and Humphreys, 1971; Oppenheimer and Connally, unpublished results; Haskett and Oppenheimer, unpublished results).
I
I
I
1
!
I
If they are, the clustering of receptors by "factor" is prob-!
ably not important in agglutination.
II
The patchy nature of the interactions at low "factor" concentra-1
tion can be explained by the presence of "factor" aggregates cross-
I
linking the cells, these aggregates being observed as the lightly
I
I
stained, amorphous material bet\-reen the cells.
Perhaps these aggre-
I
gates are large enough to interact with several receptors without
the need for clustering.
Recent work (Oppenheimer and Connally, unpublished observation)
has shown that the ascites "factors" are inactivated by phosphate
which was present in the Hanks' BSS.
This may explain the relative
inactivity of the material used in this study.
Preparations have
been partially purified in phosphate-free media which are stable
and highly active at 0.5%
v/v.
Thus the results of this study can
be considered only preliminary at this time.
Until the active com-
ponent of this factor is isolated and purified, it will be difficult
to select a likely mechanism to explain these observations.
I'
l
II_________ -. The
I
last system studied involves the use of the divalent cation
. ..... ---·- ·------ ----····-··--··· ·········-··- ····-··· .. J
34
manganese.
It has been reported that this ion stimulated the adhes-
ion of Sarcoma 1 to serum coated glass, Mn·!+ having high specificity i
i
in this system (Rabinovitch and DeStefano, 1973).
Its mode of action
is not understood, but it was suggested that Mn++ increases deformability of the cell.
More cellular projections were seen; veils,
villi, and cell flattening.
II
,
I
It must be noted these cellular changes
were seen at 1 mM Mn++ and in fact 20 roM Mn++ as used here was less
efficient in stimulating adhesion to treated glas,s.
It was suggested
that microfilaments and microtubules may be involved since adhesion
was sensitive to cytochalasin B and colchicine.
What was seen in
this study did not involve flattening of the cells nor formation of
veils.
In fact it appeared as if a stabilization occurred in the
microvilli.
Cell-cell interaction was not dependent on serum and
interactions appeared to be mediated by the microvilli.
Microfila-
ment staining appeared to be enhanced some\vhat by Mn++.
This was
obvious at high magnification (Plate l7D).
Interactions were very
close and in some cases no gap was discernible between the cell surfaces.
Ruthenium red sho\vS these interactions involved the carbo-
hydrate-rich glycocalyx of the cells and may reflect some type of
ionic bridging or perturbation of the normal ionic environment in
and around the plasma membrane.
It has been reported that lanthanium
ion can induce agglutination of Ehrlich ascites tumor and also results
in K+ efflux from the cell (Levinson, et al., 1972).
Lanthanium can
also be used to inhibit clustering of cell surface receptors (Sachs,
et al., 1974).
I
L---····-··· - · - - - · · · - - - ·-- ..
Zinc ion has been used to
11
fix" or stabilize mem-
35
r---.
..
·-·····- ·---· -·--··-----·-·-· ....... ······-···-,
i
;
lbranes (Brunette and Till, 1971).
has shmvn Zn
++
!
Oppenheimer (personal communication)
I
++
and Mn
to have a high specificity in the S-180 ag-
glutination system, and perhaps the ions are interacting with surface
glyco-protein and lipids producing a denaturation or ionic bridging
between adjacent cells.
out an
11
est in
~etermining
Mn++ agglutination may be a phenomenon with-
in vivo'' relevance though its mode of action may be of interthe nature of the cell surface.
Junctional complexes are found between many types of tumor cells.
It is difficult to assess whether or not these are functional.
As
mentioned previously, a positive correlation can be made between
greater numbers of junctions being found bet\.Jeen normal tissues than
between tumor tissues.
Also, the number of junctions correlates >vith
the form of the tumor:
for example, solid versus ascites.
All types
of classical junctions can be found in neoplasms, but tight junctions
and gap junctions are usually not as common in comparison with normal
tissues, (Trembley and Babai, 1972; Clarks, 1970; Martinez-Paloma,
et al., 1969; McNutt and Weinstein, 1969).
The existence of junctions in solid forms of S-180 has been reported (Sheridan, 1970) and these appear to be able to transfer low
molecular weight dyes and are electrically coupled.
While the im-
portance of the transfer of small molecules and electrical coupling
has not been demonstrated, it has been suggested that these play a
role in the
1973).
11
social" behavior of cells (Bennett, 1973; Lowenstein,
Sheridan's work also demonstrated that the junctions involved
in this coupling, usually considered to be the gap junction, \¥ere
I
36
··""--~·~--- ··-----~---·---~
.
~- ·------~-----·- ~-····-
. -·
---·------~--·----~---"-···
-· ..
··-·-~-----------·-
-------- -·--
;•··--~--------~----~---- -~--~------~
... -.
-------~-·~-1
!
II
able to form
bet~·Jeen
S-180 cells.
Heaysman and Pegrum (1973) examined'
the interactions of S-180 (an adherent explant of a solid form) with
chick heart fibroblasts.
Their results indicate their line of S-180
does not form specialized contacts with chick heart fibroblasts. One
might ask whether such interactions would be expected to occur between mouse and chick cells.
1,1
I
I
The S-180's used in these two studies
were different from that used here, and one must question whether
these observations are applicable in this study.
The work presented here suggests the possibility that Con A is
able to induce cell-cell associations which appear similar to gap
I
junctions.
I
with normal function, these associations could, nonetheless explain
Hhile these were not shmvn to be normal gap junctions
I
the return of "social" behavior of transformed cells after treatment
I
with lectin.
I
Ii
I
"Factor" produced interactions very similar to those
reported by Nicolson between 3T3 cells with Con A after proteolysis,
and similar to the intermediate association seen here with Con A.
This suggests the possibility that "factor"
~vorks
in a
to Con A; aggregates of factor crosslinking the cells.
~vay
similar
The mechan-
ism of action of Mn++ is difficult to assess in this work.
It pro-
duces very tight associations via the interaction of microvilli.
The significance of this in physiological terms is not clear.
i
;
i
------~J
r--------·-----~---·----------------------
........ ··-··· ..... ·--·--· .... •· ··-·-- ·····
... --------------·-- .··----· --- .·----·
--~--- -----~------1
!
Ii
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I
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..
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·~.
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r·--·-···· ---·· -------- ··- ··---··--····. --·-····- · · ·-· - ------··- -·· · · --·· ---·------...... ----·--····--·-··-----·· -· ·
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_j
l~3
1-----~----~-------~--~--=----·--·--
.
·······-·-·········-··-----------~---·--·-------····-········-·····--··········-···----
····------·---·····-···-----················,
I
Shea, S. M. (1971) Lanthanum staining of the surface coat of cells:
Its enhancement by the use of fixatives containing alcian blue
or cetylpyridinium chloride. J. Cell Biol. 21:611.
I!
)
Sheridan, J. D. (1970) Low resistance junctions between cancer cells
in various solid tumors. J, Cell Biol. 45:91.
Tremblay, G. and Babai, F. (1972) Intercellular junctions between
Novikoff hepatoma cells and hepatic cells. Exp. Cell Res. _74:
355.
Vogt, M! Delbecco, R. (1962) Studies on cells rendered neoplastic
by polyoma virus: The problem of the presence of virus related
materials. Virology 16:41.
·weiss, L. and Subjeck, J. R. (1974) The densities of colloidal iron
hydroxide particles bound to microvilli and the spaces between
them. Studies on glutaraldehyde-fixed Ehrlich ascites tumor
cells.
J. Cell Sci. 14:215.
\vessel, w. and Bernhard, \v. (1957) Vergleichende elektron. mikroskopische untersuchung von Ehrlich-und Yoshida Ascitestumorzellen~
~
Z. Krebsforsch.
62:140.
Wu, H. C., Meezan, E., Black, P. H. and Robbins, P. W. (1969) Comparative studies on the carbohydrate containing membrane components of normal and virus-transformed mouse fibroblasts.
I.
Glucosamine labeling patterns in 3T3, spontaneously transformed
3T3, and SV40 transformed 3T3 cells. Biochemistry ~:2509.
I
I
... !
Table l.
Relative Occurrence of Each Association After Agglutination.
a)
Treatmen!:_
.p.p-
Relative Percentage of Occurrence of Each Association
Microvillous
Intermediate
Close
Con A
1 hour
4 hour
60-80
60-80
20-30
20-30
5-15
5-15
llfactor 11
1 hour
4 hours
b)
10-25
10-25b)
75-90
75-90
0
0
l
Manganese ion
0' 3' 30 minutes
4 hours
Untreated
100
0
0
0
0
0
0-10
0
0
a)
The 11Relative Percentage of Occurrence of Each Association" represents a rough estimate
which encompasses both the number of times each association was seen and the relative
area occupied by that association.
b)
Microvilli appeared to be held by 'intermediate' type association.
i
i
i
j
:
i___,.__ ·---~-·--·······- ···-··------·· ··----··-----
l
-------------------··-----------····'·····--------------·-·· -------····-·--·---··----.....J
.
--- -·-· ___ , ---··--
·~
------··
--·-----~---·
--
... -----~-
·-------~-
--·· . ---· ---··-- ....,
I
l
Ii
I
Plates
45
46
Plate 1
A. Light micrographs of untreated S-180 after washing \vith Hanks 1
BSS and fixation with 2.5% glutaraldehyde and Os04. Note the large
nucleus (N) and dark staining body (1':) in the cytoplasm. 4688X.
Phase.
B. Similar to A. Note the lack of cellular aggregates. Cells appear to be mainly single cells with possibly a few pairs (--"-). 710X.
Brightfield.
c.
Light micrograph of Con A agglutinated S-180, unfixed.
large size of the aggregate. 3000X. Brightfield.
Note the
D. Similar to C, but after 4 hours in culture, unfixed. Note that
the size of the aggregate has decreased in size compared to Plate
lC.
1875X. Brightfield.
47
D
48
Plate 2
Example of an untreated S-180 cell after removal from peritoneum
and washing \V"ith Hanks' BSS. Fixation was \V"ith 2.5% glutaraldehyde and Os04. Note the irregular shape of the highly fissured
nucleus. Also the apparent emperipolesis (~). 7560X. Stained
with lead citrate and uranyl acetate.
49
so
Plate 3
A. Cells were treated with Con A for l hour and fixed in modified
Karnovsky' s fixative and Os04. Micrographs shmvs a nuclear caniculus (c) associated with the nucleolus. Also note nuclear vesicles
(-) and unusual aggregate of tvhat could be viral cores (.-..). Pars
amorpha, pa; Pars granulosa, pg; Pars fibrosa, pf; chromatin, Ch;
nuclear pore (.-.). 24,600X. Stained \vith lead citrate and uranyl
acetate.
B. Cells treated as in A. Note two large nucleoli, nuclear caniculi (c), and cytoplasmic channel (~) associated with one of the
nucleoli. 17, lOOX. Stained with lead citrate and uranyl acetate.
51
52
Plate 4
Cells treated with "factor" for one hour and then fixed in modified Karnovsky' s and Os04. Plate shmvs centrosphere region, note
Golgi apparatus (G), and numerous vesicles and vacuoles in the cytoplasm. The centriole (Ct) is prominent and so is the large nucleolus (n). Nucleus (N), mitochondrion (M). 42,600X. Stained
with lead citrate and uranyl acetate.
53
------~--------------------~-------------·--·--·---~
-
54
Plate 5
A. Cells treated with Con A for one hour and fixed in modified
Karnovsky's and Os04. Note the Golgi apparatus (G) with large
number of associated vesicles and vacuoles (v), the dense staining body(~), endoplasmic reticulum (E), and autophagic vacuole
(av). 25,400X. Stained with lead citrate and uranyl acetate.
B. Cell treated as in A. Plate shows the condensation of several residual bodies with an autophagic vacuole. 49,000X. Stained
with lead citrate and uranyl acetate.
C & D. Untreated cells fixed in modified Karnovsky's and Os04.
Shows a large lipid droplet in the cytoplasm and the apparent exocytosis of a lipid droplet. Mitochondrion (H). C. 8560X. D.
8320X. Stained with lead citrate and uranyl acetate.
E. Cell treated as in A. Note the large dark and irregularly
stained bodies, and the myelin figure (-+).
13650X. Stained with
lead citrate and uranyl acetate.
F. Cell treated with factor for one hour and fixed with modified
Karnovsky's and Os04. Example of another type of dark staining
body. 40,800X. Stained with lead citrate and uranyl acetate.
I
55
56
Plate 6
A. Cells treated with "factor'' for one hour and fixed in modified
Karnovsky's and Os04. High magnification of virus particles in
cytoplasm. 114,660X. Stained with lead citrate and uranyl ace-·
tate.
B. Untreated S-180 cells after \vashing with Hanks' BSS and fixa..,
tion with 2.5% glutaraldehyde and Os04. Note the variety of surface projections shmvn by the two cells and the lack of interaction
bet\veen the cells. Apparent emperipolesis (\/). 15,700X. Stained
with lead citrate and uranyl acetate.
C. Cells treated with Con A for one hour then fixed with modified
Karnovsky 1 s and Os04. Shmvs the interactions between normal (Y)
and an apparently dying cell (Z). The obliqueness of the section
does not allow a clear determination of the intercellular area.
30,000X. Stained with lead citrate and uranyl acetate.
57
58
Plate 7
A.
Cells treated with Con A for one hour and fixed \vith modified
Karnovsky's and Os04. Note the large number of microvilli which
have been agglutinated to the surface of the parent cell and also
the number of cell-cell interactions bet\veen the microvilli and
other membrane projections. 18,100X. Stained with lead citrate
and uranyl acetate.
B. Untreated cells fixed with modified Karnovsky's and Os04. Compared with A, the microvilli tend to project from the cells and
are not flattened to the cell surface; also the number of cellcell contacts are reduced. Mitochondrion (M).
18,100X. Stained
with lead citrate and uranyl acetate.
59
60
Plate 8
A. Cells treated with Con A for 4 hours and fixed with modified
Karnovsky 1 s and Os04. Plate shows an aggregate of five cells.
Several types of associations appear to be involved. Note that
the microvilli in the free regions are apparently projecting out
from the cell, and are no longer agglutinated to the surface.
Intermediate association(-), close association~).
15,650 X.
Stained with lead citrate and uranyl acetate.
B.
Cells treated as in A. An extreme example of the extensive
interactions which can occur bet\veen villi of adjacent cells,
Note how adjacent cell villi are pushing into the cell surface of
the other cell (~) and that some of the villi appear to branch
( +). 35 ,400X. Stained with lead citrate and uranyl acetate.
C. Cells treated with Con A for one hour and fixed with modified
Karnovsky's and Os04. A region in which villi are pinned between
adjacent cell.s. Note some villi are agglutinated to both cells.
A close association is also seen in this region (-).
10,300X.
Stained with lead citrate and uranyl acetate.
61
62
Plate 9
A.
Cells treated with Con A for one hour and fixed in modified
Karnovsky's and Os04. Note lightly stained material occurs only
in region where intermediate association (--) occur. 68,000X.
Stained with lead citrate and uranyl acetate.
B.
Cells treated with Con A for 4 hours and fixed with modified
Karnovsky's and Os04. Region of interaction between two cells in
which intermediate associations (bars) and microvilli interactions
(-+)occur.
21,900X. Stained ~vith lead citrate and uranyl acetate.
C.
Cells treated as in B. T~vo cells held by several small intermediate associations (-).
22,300X. Stained with lead citrate
and uranyl acetate.
63
Plate 10
A. Cells treated with Con A for one hour and fixed in modified
Karnovsky's and Os04. An example of the close association seen
after agglutination. 203,000X. Stained with lead citrate and
uranyl acetate.
B. Cells treated as in A. An example of a common type of association, probably a close association cut obliquely in several
regions (--). Note villi between cells. 37,300X. Stained lead
citrate and uranyl acetate.
C. Cells treated as in A. This shows adjacent cells held by an
extensive close association. 68,000X. Stained with lead citrate
and uranyl acetate.
D. Cells treated as in A. Another example of an extensive close
association.
54,600X. Inset illustrates the close apposition of
adjacent membranes and also shows an unusual thickening of the
membrane which appears to be a third bilayer (-H-). 22l,OOOX. [ ]
region enlarged. Stained with lead citrate and uranyl acetate.
65
66
Plate 11
Cells treated \vith a high concentration of 11 factor 11 for one hour,
then fixed with modified Karnovsky' s and Os04. Shmvs the extensive pinocytosis and phagocytosis of the flocculent material on
the cell surface. About 7500X. Stained with lead citrate and
uranyl acetate.
67
..
r
.
I
68
Plate 12
A. Cells _treated >vith a high concentration of "factor" for one
hour then fixed with modified Karnov~ky's and Os04. Higher magni·fication of the flocculent material on the cell surface. 60,000X.
Stained with lead citrate and uranyl acetate.
B. Cells treated as in A. Note the large amount of material adhering to the cells' surface. Interactions seem to be the result
of the material on the cells' surface (~). Also seen are numerous smaller lysosomal vesicles (y) possibly GERL (Novikoff, 1973)
which appear to fuse (--)with the larger residual body (Rb).
13,700X. Stained with lead citrate and uranyl acetate.
c.
Cells treated as in A. Shmvs the large amount of material
sticking to the surface of the cells. Interactions appear to be
due to the material on the cell surfaces. Again numerous lysosomal vesicles are present (y). Note the swollen ER (E) filled
with a moderately stained material. 20,000X. Stained tvith lead
citrate and uranyl acetate.
D.
Cells treated with a low concentration of factor for one hour
then fixed \vith modified Karnovsky's and Os04. Shows the exocytosis of two large myelin figures. It appears as if the cells are
being forced apart by the vacuoles. Note the surfaces of the adjacent cells appear to be complementary. Several autophagic vacuoles (av) can be seen in the cells. 20,000X. Stained with lead
citrate and uranyl acetate.
69
70
Plate 13
Cells treated \vith low concentration of "factor" for one hour then
fixed with modified Karnovsky's and Os04. Note the numerous villi
and lack of vacuolization as seen at higher concentrations of
"factor". Also note viral aggregates (V) in the cytoplasm. About
7000X. Stained with lead citrate and uranyl acetate.
71
72
Plate 14
A. Cells treated for one hour with "factor" at lmv- concentration,
then fixed with modified Karnovsky's and Os04. Sho-.;vs region of
intermediate association (rt); note microvilli involved(--).
23,600X. Stained with lead citrate and uranyl acetate.
B. Cells treated as in A. Sho-.;vs patchy nature of the interaction
bet-.;veen these cells. Bars indicate regions in which patchy material occurs. 23,700X. Stained with lead citrate and uranyl acetate.
c.
Cells treated as in A. Sho-.;vs region of intermediate interaction. Sl,OOOX. Stained -.;vith lead citrate and uranyl acetate.
D. Higher magnification of A. Note the interaction appears to be
mediated by patches of staining material (-). 65,400X. Stained
with lead citrate and uranyl acetate.
73
74
Plate 15
A. Cells were fixed immediately after addition of Mn++ with 2.5%
glutaraldehyde. Note the lack of large aggregates. 600X. Phase
microscopy. Postfixed with Os04.
B. Cells were fixed three minutes after addition of Mn++ with 2.5%
glutaraldehyde. Shmv-s a large aggregate of cells.
1500X. Phase
microscopy.
Postfixed with Os04.
C. Cells were fixed 30 minutes after addition of Hn++ with 2.5%
glutaraldehyde. High magnification of a small aggregate. Microvilli can be made out in areas. 3750X. Phase microscopy. Postfixed with Os04.
D.
Cells were fixed four hours after addition of Mn++ with 2.5%
glutaraldehyde. Note vacuolization of cells, also no aggregates
are seen.
1500X. Phase microscopy. Postfixed \vith Os04.
75
c
76
Plate 16
A. Cells were treated with 20 mM Mn++.
2.5% glutaraldehyde was added
immediately after addition of Mn++. Shows the interactions between
the cells are mediated by surface projections. Note that interactions are not very extensive.
7000X.
Postfixed ~vith Os04. Stained
'vith lead citrate and uranyl acetate.
B. Cells treated as in A. Shows interactions can occur between several types of surface projections. 12,500X. Postfixed with Os04.
Stained with lead citrate and uranyl acetate.
c.
Cells were treated with 20 mM Mn++.
2.5% glutaraldehyde 'vas
added after three minutes. Shows that the number of villi involved
in interaction has increased and some cell flattening has occurred.
14, lOOX. Postfixed 'vith Os04. Stained Hith lead citrate and uranyl
acetate.
D.
Cells treated as in C. Villi interact at point contacts. The
cell surface does not smooth out in the regions of interaction.
27,300X. Postfixed with Os04. Stained with lead citrate and uranyl
acetate.
77
78
Plate 17
A.
Cells treated with 20 l11i'1 Mn++ for 30 minutes then fixed wHh
2.5% glutaraldehyde. After extended incubation, interactions are
still similar to those of shorter duration.
17,100X. Post-fixed
with Os04. Stained with lead citrate and uranyl acetate.
B. Cells treated as in A, but fixed using the ruthenium red
staining method. There appears to be no differences in staining
properties between those areas of interaction and those not involved in agglutination. 38,200X.
c.
Cells treated \vith 20 mM Mn++. 2.5% glutaraldehyde Has added
immediately after addition of Mn++. Plate shmvs the interactions
168,000X. Postfixed \vith
are tight between villus membranes.
Os04. Stained with lead citrate and uranyl acetate.
D. Cells treated with 20 l11i'1 Hn++ for three minutes then fixed
with 2.5% glutaraldehyde. Again villus interactions are very
tight~JPqte also obvious microfilaments in the villi (--). 226,000X.
Postf~ed with Os04.
Stained with lead citrate and uranyl acetate.
?r . ,
..,.,
>
'J
79
80
Plate 18
A.
Cells were treated with 20 rru'1 Hn+t- for four hours, then fixed
with 2.5% glutaraldehyde. Cells have become very vacuolated.
Cytoplasm seems to have two distinct areas, one staining darker
(d) than the other (1). Mitochondria stain darkly. Villi are
much reduced in number.
7000X.
Post fixed \vith Os04. Stained
with lead citrate and uranyl acetate.
B. Cells treated as in A. Unusual cell with large number of
vacuoles and completely smooth surface.
7800X. Postfixed with
Os04. Stained with lead citrate and uranyl acetate.
C. Cells treated as in A. Cell has villi and is not vacuolated.
Cytoplasm appears to have a dark and a light staining region.
3510X. Postfixed with Os04. Stained \vith lead d_trate and uranyl
acetate.
81
A
.B·