CALIFOHIEA STAT:-:-: UNIV'::nSI'J.ry, NOH'P?FUDGF
T'HE iiDH:ss ION OF SEA urlCEIN ·E':VIBRYCNIC
CELLS TO PULYSACCHARID':: :g::ADS
[\ tlws:i.s submitted in partial satisfcwti:""Jn of tb.'-';
r6ou:l.rements .f'or the degree of [·;laster of ~~cit:n~ce ~~.n
Bj_ology
by
Gary Paul Toedter
~-
..Tune ,
19'78
The Thesis or Gary Paul Toedter is approved:
Date-
Cn.lif'ornia Sts. te University, Northridge
ii
A BS 'TH it C'.l1 ~
a • •
•
lNTHODUCTION.
• •
e • e • • • •
• • • e • • • • e ~ • •
YiA ~f'EH IA IS AND
I~n bry o s
• e • • • • • • • e • • e e
• "'
8
o ••••
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iD' . . . . . . . .
6
e
~
e •••••
o
8
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e • • a o •
.v
• • • • • • • • • • • • • • e
• G •
G
•
6
e • e e e e & • •
o • e
.3
..........3
••••
Dissociation •• o~·•••••••••••••••••••••••••••••••3
.ilf:f~I~G[~atj_on l'1.ssa-y·se •
~'canninG
c • •
~
• •·•" • • •"' o e • • • • "e •
}t;lec tron Microscopy.
Electroptoresis ••
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e e e •
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* •., • •
• • • e
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•• 5
Cross-linking and Reduction of Bead Surfaces •••
.,s
Dete:c'mination of Charge on Bends ••••
•
P erioda te Treatment ..
R. ~~;:~ uLrr s-a
v ._.
II
••••••
e ••••
Q
Hapten Inh:i.bition.
•••
ft
~
•
Other• Composition Beads ...
DISC T..;s S I 0 l'l{) •••
Cl
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e s
€>
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......
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ll
.11
... .... .. • 1.3
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Cl
..
~~
.............. •
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'ft
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... s
& ...............
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r,
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R~~~??ER}~l'JCE~3~ ....... ~ ........... "' •••• q"' ••••
~liGUR-ES. • • •- .... • • .. • • • • ~" • • ..
•
& 0
0
Cress-linking and Reduction ••••••
e
•
0
0
of' Glycos idas es.
Other Procedures.
•
•
•
Scanning Electron Microscopy.
Perioda te Treatment ........
•
•
••••
• • •* •
•
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&
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e .. • • •
2~~
24-49
LIST 01•' FIGUR:C.S
1,. Diagram of electrophoretic apparatus ....
2.
~ffect
of monosaccharides upon
~~
.... .,.24
adhesione•~···26
3Q Effect of galacturonic and glucuronic acid
upon adhesion •• o•••••••••••••••••••••••••••••28
4. Effect of divalent cations upon adhesion ••••• 30
5~ Effect of 10~ 2 M
cac1 2
upon adhesion •••••••••• 32
6 .. Adhesj.on to beads other than Seohaross •••••
~.34
Initial appearance of cell-bead clumps ........ 36
S~
9~
Light microscope view of cell-bead
ag~regste.38
Scanning electron micrograph of cell-bead
aggregates•••••••••••••••••••••••••••••••••••40
10. Cells and beads in M?-SW ••••••••••••••••••••• 42
lL Destained gel on wh5_ch clycosidases v.·e1'e run .. 44
12~
Effect of a pH gradient upon adhesion •••••••• 46
13. Effect of a ,pH gradient uoon adhesion to
D.EAF;·-Biot~el
•••
li'
•••••••••
i-v
.,
••••••••••••••••••
e
.,48
ABSTRACT
'rl1E i-LCEESION OF SEA URCHIN
T~'MBRYCNIC
by
Gary Paul Toedter
Master of Science in Biology
Dissociated sea urchin embryonic cells
w~re
observed
to adhere to Sepharose 4B beads when the cells and beads
wer·e rotated together in calcium-and-magnesium-free sea
watoro
When placed in Millipore filtered sea watsr, the
'
cells would not adhere to the beads.
It was sbown that
both calchJJn and magnesium ions could separately inhibit
,
.
ao.n.es ~on.
t;J:J;
Galacturonic and glucuronic acid increased
adhet-:;ion, while ether haptens had no effect.
'l'he ee1ls
would a.dhere to other comoosition beads, Bnd could he inhi b:'Lt ed hy the add it ion of' calcium or magnesium ion.
It
was also shown that the adhesion to Senharose 4B was pH
dependent.
Various mechanisms for explaining the adhesion are
v.
discussed.
Chnrge-charge, hydrogen bonding, and van der
Waals interactions are possibilities.
The cell-bead interaction observed here can serve as
a model for demonstrating initial cell-cell interaction,
leading to specific cell-cell adhesione
vi
INTRODUCTION
Studies on the interaction or cells with artificial
substrates may yield important infor·mation as to the nature of cell-cell and cell-matrix interaction-in vivo.
The adhesion of cells and dissociated tissue to inert substanceo has been studied by J·acobson (1978) and
( •og•-;t=;\
l..
'
i
'L'
c
~i~azia
These investigators used polylysine absorbed to
glass beads to provide a positively-charged
surface~
Jacobson (1978) faun( that cells adhered tenaciously to
the bead surface to such an extent that a
nortio~
of the
cell was left attached to the bead after the cell-bead
- J.na
•
t_; 1.0
• n
c or:m
\~'~J8.s
sonicated.
adher~
Chinowsky et al ( 1973)
ed cells to SeDhadex beads to vrhich they had previously
bound sugsrs covalently
~y
arm att2ched to the sugar.
the use of a molecular
spac~r
This paper presents similiar
work in whi.ch dissociated 24 hour sea urchin embryo cells
are obServed to cling to untreated Sepharose and other
polysucchuride beads.
It was observed initially that
S~pharose
beB.ds, when
added to a suspension of sea urchin cells, adhered to the
c e l lB only in calcium-and -mar;nes :Lum-fr e e sea water.
The
cells and beads would not adhere when placed in n.o.tural
sea water·.
This vTas thought to be contrary to Eumphrey's
(1963) observ~tion on the necessity of calcium ions for
1
2
intercellular adhesion (non-bead, however).
The cell-
bead interaction was investigated to determine the cause
or the interaction in terms of possible
c~rbohydrate
in-
volvenHmt (Roseman, 1971), end possible non-specific
cbBJ:'ge phenomena.
'l'he role of calcium and magneslum ions
in inhibition was examined also.
that inter•actions such
~aals
HS
hydror~en
These results suggest
bonding and van der
interactions may be imnortant in cell-bead binding.
gs and sperm were obtained from the sea ur-
chhw ~-··.J.~....
Lvt echi:nus -oic
tus
and Stronr-::r
loc en
trot
us purmJ.r·---""'*'"'
.
- "'
~~.by
injections Y'ith 0.5M KCL
The
ec~gs
were washed
three times with Millipore-riltered-sea water (M?-SW)
and fertilized.
1.'-'"'n·,-,
LrJ ._,_.. l
After the fertilization membrane had
(r,•)n""ox·l··na,l,.nlv
<15 secol:1ds)
c:.:.... t ~.
.l.lc L, ....~ v
J.i.
_
.,l..
•
'"·
-.·
'
Z'-rn·ote"'
"···re
dJ"lu
.J;:- C;
-'~::>
\/;' t::
.
-
·t·l.-,r_,
1.i.lV
'J
(10
'•TP-C''!'
....
0,1
~
incubated ;::.t 17°C in pl&stic petri dis'r,es tor
DIS~~.OCIATIOIT.
Embryonic cells
method of Opper::heimer ( 1973}.
dissocliJ.tc:'d b:y the
V."Gre
'r.J'nbryos v,·e:re ,,;ollected by
lov; speed c entr ifu::a tion, washed three t
p·ncJ-n,a,,·rl""S.ium-Pree SPa W8ter
... ......
.........
ethyl
.__:;
......
•
...
-~-
~C;ther)N,:N
St& Louis,
J
•
1'
~o.),
and the c0lls
·c..J
•
(r.:.,,~~-O,'r!)'
' - ' - .......
..__
• '
'
irnc~s
5.n CBlc i1.lrJ1··
rt3E-usoe·r1.o.'ed ir:1. C'/';-:-
.
-tetra-·acetic acid (S:ir;:mR. Cherrdcfl Co,
0.02M Tris at pH 8.2,
d~ssociated
~or
ten minutes,
by gently pipetting.
Cells
were diluted with 10 mls of CMF-SW 1 centrifuged, and resuspended in c~rP-SV!, ccntaininr~ 10 ug/rn1 DNAase to inhib~
it art ifrc.c tua 1 agp:lutinat ion crn.1s ed by DNA.,
out .in 1 dram vials, cont:;dning 1 ml of CI\'IF'-SV! in v1hich
either a rr,onNJaC char ide, sp cc if i.e glyc os idas e, diva lsnt
4
cation, or other test substance was dissolved.
One-tenth
of a rnl of cells (150,000 cells/ml) was placed into each
C~F-SW
vial with the
solutions.
The vials were rotated
on a gyratory shaker (50 rpm, 4 5/8 inch diameter of ~otation, Oppenheimer, 1975) for
either 0.1
~ls
r:::
0
•
....
mlnUueS
•
At this point
of Sepharose 4B beads (Pharnacia, Piscator other snecified
beads~
were added and the viats rotated for 5 additional minut()B.
4.
~n.lc
The contents of the vials ':·'ere then dil-u.ted with
of CMF-SVl and filtered th:roough disposable glass
Pastetrr· pipettes
containir~g
in the neck of the pipette.
loosely packed rayon fibers
It was shown that the rayon
fibers effectively prevented the passage of the beads
(or beads with attached cells) but allowed single cells
to pass throue;h uninpeded.
The filtrate vms then diluted
to a total vo lv. me of 10 mls, and the
the filtrate
det~rmined
~mmber
of cells in
with an electronic particle coun-
ter (I.. Iodel ll2LT Celloscope, Particle Data, Inc., Elmhurst, IlL,, current-l/2, r;ain-24.).
An increase in the
nu1nber of eells (as compared with control vio. ls) corrGf>·
ponded to a decrease in adhesion, and conversely, a decreaE;e in the number of cells corrJsDonded to an incroaPe
in adhesion to the beads.
Vials were checked
visuall~
bGfore filtering to check for visible clumps.
bility was detersined by Trypan blue exclusion.
Cell via-
SCANNING
E~_T:C'l RON
1
N!ICROSCOPY.
Beads with cells attached
were .fixed in 3%' gluteraldehyde in O.lM phosphate buffer
(pH 8.2} containing
116
sucrose, for 24 bourse
cell conmlex was then wa:Jhed in
in 2fo Os0 4 for 2-4 hours.
bu~'.fer,
'I'te beBd-
and ::)ost-.f'ixeQ.
A:Cter being washed in buffer,
the beods and cells were dehydrated in a graded se·--ies of
ethyl alcohol} and critical -:::-;oint driec' with carbon dio .xi d e in a P e 1 c o T:1 o d e l B Cr i Li c a l Po in t
Dry er •
'l' he c ell-
bead como lex was coat eel with gold-pa llad iuin, and examined
i~
an ISI Super Mini Scanning electron microscope.
Chemical Cmllpany were tE;sted for p-u.r-ity by slab gel
cont::l_nous electropl·oresis.
dis~
A lC~t polyacrylimide gel,
Richmond, CA) was used as the separating
pH 6o7 gel used for stacking.
Ten micro-
grams o.f each sam le vvere run simultaneously vvith 100
volts, 120 mill:l.amps aeplied to the unit for 2 hours.,
The slabs \':ere stained in Cornass i e Brilliant Blue and
deL~
stained with a 10~ acetic acid-1~ methanol solution.
The slab was photogranhed, and nurity was shown by the
appearance of a single band.
CHOS::'-LTlEING AND R-:-'l)UCTION C':' :FAD
SUP.FAC:::~s.
Sepharosc
4B, Sephadex G-200 (Pharmacia) and 200-400 mesh Bio-Gel
A-l.5m, were treated according to the method of Porath,
e~.
al ( 1971).
Tbis method attempts to neutralize the
su.rface charge .fotmd on the beac's by cr'oss-linking and
6
reduction of bead irrpurities.
surfa~e
The
charge is due
to 5_nrourities in the (Jesds in the form of car'oxyllc and
sulphate ester grouos.
T~enty-~ive
mls of washed beads
wore mixed with 25 mls of 1M NaOH containing
epi(:hlorohydrin
(vv:n
h(·,·urq
~-
1-'
•u'tth
~. ~·
J,..L
mls of
Scientific, Los Angeles, CA) and
1~25 gms sodium borohydride (rt.C/B,
room temperature.
2~5
Los Angeles, CA) at
The mixture vvas he[lted to 66° for 2
occ~R~onal
C~r.....:: -'·.
•
st iJ?ring.
The gel was then washed
with hot distilled wster until the Gel suspension was
neutral, and the bends autoclavod for one hour in a mixtu:re of 2]';1 NaGH (15 mls) End 0.6 gms sodimn borohydride.
'I'he gel
1N&s
then washed with '75 mls of hot 1M Nao'-r, and
75 mls of cold lM NaOH.
a beaker
conta~ning
The gel was then transferred to
crushed ice and acetic acid was added
until the susnension was pH 4.0.
The gel was then trans-
fer:;:oed to a Buchner f"Lmnel and v.cashed with hot distilled
water to
ror:~ove
any remaining boric ecid and then with
cold distilled water~
frigerator.
The gel wrc:,s stored in a 4°C re-
This treatment was designed to remove the
sulphate p;rouc-s by alkaline hydrolysis, and to cross-link
tho
C~:!.rbo::;dlic
groups with the epichlorohydr:i.n.
This is
PI'esuJnod to produce bes.ds with a reduced charc;:e.
Dl:,T~'J{'\UNATION
OF CEP_Rr;E ON IFADS.
S'he charge present on
the beads wr::.s determined qualitatively by electrophoresis.
An electrophoretic app~ratus was constructed (Figure 1)
from
plexiglas~.
The main chamber of the apparatus was
'7
filled with glycine electrode buffer (p"Cf 8.3) containing
15% sucrose (wt/wt).
The two side chambers held conper
electrodes and v;ere filled with the electr·ode buffer.
The electrode chambers were separated from the main chamber ·by dialysis tube split lengthwise H1d clamped between
the arms of the two chambers.
The bottom of the apnaratus
we.s seale6 by placing the unit in a shallow nlexiglass
u~polymerixed
trAy filled with
mc:.cization, the gel effectively
acrylirnide gel.
se~"-led
Upon poly-
the unit.
o:r sus·.·; end sd beads was ca;oe.fully c) laced upon the
1\. dl~op
buf_~~
er
in tha main chaiiiber and allowed to ff-i 11 between the two
eleet:r'odes.,
r;he path
o~·
the beeds 'i'as tracked as a
rent of 250 volts was appl1ed.
char~e
meant a net necative
cur~
A de.flection to the anodcJ
existed, and a movement to-
ward the csthode meant a net positive charge.
The 15~
wt/wt sucrose was used so as to slow down the speed at
which the bends fell.
r
SO:::HU~/I
P~:-.-?IODA'""=:
TP:~Tl·:I'-;:N'r.
The erfect o.f sodium nerio-
date treetment of the cells upon the cell-bead adhesion
wafj
de's'3rDined.
Cells were fixed with 3% f'orm8L;ehyde,
washed three times in C~F-SW, and incubated with 10-lM,
r;
end 10- 0 M sodium period<: te
J'-,1F-SW for one-half hour.
bated with periodate.
c· •
'
( ._olgma;
•
ln
Ctil"P-SVV and
Unfixed cells v1ere also ineu-
Sepharose leads were then added and
the beads and cells rotated for five additional minutes.
The cell-bea:Js ·were
a~sayed
as in the previous m8nner.
RESULTS
Many di.L'erent monosaccharides were
Hl\.l>T:rm JNEIBITIO::\.
used in an attemnt to inhibit the binding of the cells to
implic8f~e
the bends and thus
in the adhesione
a specific tern:inal residue
D-Galactose, D-fucose, D-mannose,
D-f'ructose, D-fucose, D.-xylose, N-acetyl-D·-gluccsam:Lne,
·' .
( a1., l from Sigma) were
D -r1~ose,
an d D- ana' L -araolnose
these
SlJ;~ar.s
ht::.d an
e.~'fect
on the s.dhesion, eithe:r• in B
positive or neg<?,tive manner (Fi :ure 2).
However, D-t;luu~:tcma)
co:::;e anJ the Bcid sugers, D-gB.lacturonic acid
and
D-glucu:r.•onic D.cid (Sigma) did affect the adheslon.
D~Glucose inhi'~ited the s.dhesion at 10-lr.i (a 10% reduc-2
tion in adhesion), and at 10 lv1 it caused a 5?S ruduction.
D-Cialactur'onic and D-ghJ.Curonic acid affected the adhesion
in a positive manner (Fizure 3).
At pH 8.2, salactur-
ordc acid (pKa 3.42) and glucuronic acid (p1\:a 3.2C) are
nesati vely chs.rged.
DIVJ~L:~N'I'
CA'l'I:<I::s.
Calcium, magrlesium, zinc, and mangan-
ese ions (all in the
chlo~ide
form), when added separate-
ly to the CMF-SW containing cells, had en impressive e.ffeet u:oon c e 11-bead odhes lonG
10
-1
'Z
·Iii to 10- 0 M,
Prom cone entr' t ions of
calcium ion completely inhibited adhesion
of the cells to the becds
(~igure
4).
At 10
-Ll
~M
this ef-
9
feet was somewhat less.
Normal sea water has a cat+ con-
centration of 2.9 X 10=2:·:1.
Thus, calcium ion is inhibit-
or·y at concentrations equal to or less tEan are nor:mally
I''ound in T·:i?-SW.
Strong catr inhibition vras further demon_0
strated by the nddition of 10 "'y calciu..-rn to cell-bead
clumps ..
'rhe clumns vvould begin to fall [-,_part immediately,
and by five minutes were more than 25~ dissociated (Fig-
ure 5)
Magnesilun ion is inLi'oitory also; e.lthough to a
mueh lesser extent than calcium (?'izure 4).
The
:r·rg++
ions at concentrations from lo-lm to 10- 3 M inhibited ad,, 1)01)-l-
c--.."-
.u
0
~
0 .·"•
cf.
concentracion
higher• than calcim:'!, being 1.5 X 10-l;':T.
Manganese ion has a
~ositive
effect on the adhesion
is present in sea we.ter only as
4:) •
a trace element.
Zinc chloride for:ms a heavy white preci"8itate -vvhen
(
added to CLIF- s>::, thus preventing 2.ny testing of its
ef~
f'ec t.
O'I'HT,::R
co:.·'~CSITION
:::~-;:~·Ds.
In <i.ddition to Sepharose 4B,
cells adhered to the surface of polyaerylimide beads
(Bio-Gel ?-100), diethylaminoethyl
(D~Jl:-=:)
agarose (Bio-
Gel) , carboxyr:l8thy 1 ( Civi) a::;aros e ( Bio-Ge 1) , Agaros e ( BioGel), Ultrogel AcA22 (LKB, Franco}, and Sephadex G-200
(Pha.rmacia)
e
l'/Ioreover, the
adh'c~sion
of the eells to the
beads was inhibited by calcium or MF-SV! in the Sb.me man-
10
ner as the Sepharose-cell interaction (Figure 6).
SCAYNii:TG
'~LC:::C:l_1RON
T'IIICROSCOPY.
VJhen observed f'loo.ting in
a Petri dish, the cell-bead clunms appear as in Figure ? •
They form
8
m~ws
of cells o.f' various shspes, usually
round and l em in diameter.
When observed with a light
microscope, the beads can be
distin~uishcd
(Fig1J:ee 8).
from the cells
The cells cs.n be seen to stick to mor3 than
one bead, thus fo1'r:ling the mass seen beforeo
bead
clw~p
as seen with the scann1ng electron microscope
Jp illustr·ated in Fi[11re 9.
what
'Jlhe cell-
dama~ed;
'l'he cell's surfnce is some-
apparently by the dissociation treatmento
The cells cling to the beads, and also to each other.
A
large percentage of the cells, when attached to the beads,
show this cell-cell interaction.
The cells adhere to
each other only in the presence of the beads.
A view of' the cells and beads ss they W'pe;::;.r in NIP··SW
i s s e en in ?i cure 10 ~
'-·-.:c:-:_:c•TROl'FOW:O:SIS OF
GLVCOSIDAS...,.~).
The purity of the gly-
co::noas es obtained from Siona was determined by elHctrophoresj_s, ·with purity being presuined by the app::wrance of
a single band.
B-Galactosidase (G-8504) and alpha-gal-
actoside,se (G-900?)
showed only one band (Figure 11),
and when these two enzymes were used,
they showed no ef-
fect upon the adhesion of the cells to the
P:?IUODJ',J:E
'I'R'-=~AT;,ErFr.
beads~
· 1-:1eriodate treatment interfered with
cell-bead interaction.
Live cells showed a
dec~ease
in
11
cellular adhesion to the beads.
In CMF-SVJ, the a verap;e
nurnb0r of periodate-treated cells decrucsed by a1)proximately 90?t as compe.red wit~l live cells in CMF-SVJ.
Treat-
ing fixed cells with periodate did not affect their
hesion~
ad~
J:ll1is is born out by the observation that NIF-S'-N
1
did not inhibit the adhesion in the usual fashion.
This
does not imply that fixing the cells -;;revents the inhibitory action of
ca+t
and
Mg++
ions-rather, it shows that
fixing reduces the adhesion of the cells to the beads.
tion o:" tb.e boads was carried cut in an attempt to I'E-;duce
or neutralize the charge present on the beads.
The beads
were electrophoresed to determine if the charge on the
beads wcs
t.reP.h:;ent.
a~fected
by the cross-linking and reduction
By v:i.sual observation of l;e8d rnovGment in the
electric field, the trePtment did not affect the charge.
Bio-Gel A and Sephadex G-200 moved toward the anode, as
t
did untrected Sepharose 4B.
were run as controls.
and
D~A::::-Biogel
D':'AE-Bioe;el and CM-Biogel
CM-BioGel moved toward the anode,
moved towe.rd the c2.thode.
The
cross~
linking FJ.nd reduct ion of' Sepharos e 43 was unsucc es srul,
as Senharose melts when subjected to 120° in an autoclave.
O~JlH~:R
PHOC ·:DUHES.
The effect of pH upon sdhosion vvas de-
terrnined by pl&cing the cells and beads to7ether in l ml
of CNIP-SV,' with. the pH varying from 4.0 to 10.0.
As can
s.o,
be seen from Fic;ure 12, adhesion decreased below pH
reaching a low at pH 6.0.
At pH 9.0, adhesion is near
norrnal, and at pH 10, was slightly above normal.
By contrast, the pH gradient cave differeLt results
using
DEA'~-Biogel
instead of
Sepharo~~e.
At pH
s.o,
a
decrease in the number of free cells appears (Figure 13)
A. dscrease in cell-'oead adhesion appears at a:)out pH
5.0.
CI':t-Biogel gave inconsistent results when utilized.,
Q
DISCUSSION
When first observed, the adhesion of the cells to
the beads vn:Js reminiscent of the results
powskJ,
~!
al (197;3).
obta~ned
by Chi-
Br•iefly, whs.t they observed was
the adhesion of c~ltured fibroblasts to Seohadex beads.
The beads had various specific
attached to their surfaces.
~usar
residues covalently
There are three
im~ortant
differences b";tween the observations of C!::inov:sky, et al,
c
-
...... -.....
and the present observations: 1) the Sepharose 43 used in
these experiments was off the shelf; it has no deliberate ly Ettach:::1d sue st.snc es.
Chi~)mvsky
et a 1 saw no nttach-
ment to their untreated control beads, 2) the strong cal-
c ium inhibition observed here was not seen by Chipov:sk:y
and his
co~orkers,
and 3) all of the cells would adhere
to the beads within five minutes in the present work.
The cells would take more than one half hour to
adh~re
to
the beo.1ds in the exper_iments of Chipovrsky, et al.
The cells and bee.ds form large clumps
rotated to:;ether.
a.~·b:r
being
The cells adhere tens.cim:sly to the
beads; which is evident by their resistance to being
broken up by gentle pipetting.
The lack of inld. '~~i tion by the haptens used suggest
that cell-bead adhesion is not sugar specific.
If a su-
gar residue was involved, then the
ad~esion
misht have
been inhibited by the addition of specific SUfars.
This
ro llows the line of reasoning of Por3 eman in h5_ s galee tosy l
+I'8'
. ri.iatvc.:o'
V
~ ..JRr_prc~.~-".1P
·--"
~-·~
·-·
-•
r~hos10r1
--
c.__,,~·
0 J'S
~VDOthP
Eu ,··
v ___ ,~,
·•
>.,
(Rc·J"Ow~n
' ' . C'J ;I.iC'.
1
QY~/ -'S
..t..v
"'
I·L)
6
In addition, the use of specific glycosidases showed no
signifi~ant
are
effect unon adhesion.
comno~~ed
of poly[:';alactose
un:tt~"J
Since Senharase beads
9
s·9ecific inteP!':ctLGn
vmv.ld more tr:an l.ll<ely involve galecto:.Je (or a s:1::-nilsr
nnd a galactosidase micht h;:--,vo
lnhibi~~t~d
the ::.tdhes:lon.
The app<>r'er..t inhibition of adhesion '!:Jy glucose is
d:i.ff~~C't.l.lt
to GXPl&in.
11 his inJ:-libition vvoulc seem to in-
dicate a specificity in the adhesion, but as the cells
ndl':.er e to dlff erent ty;J es of beads, this is unlikely e
Seph2.ros e 43 is com. ,os ed of ga lac toPe ( a lineaJ:.' po lyJner•
Rosen, 1973).
Sephadex G-100 is com~osed of dextran (gluf
case nolymer, cross-linked at carbon 3, (Morrison ard
l?.oyd, 1974) ~
catF.t 1.og
'J
Bio-Gel F-100 is polyacr::'U.mide (Bio··'Re,~
:-1r1 d Agarose is D- and L-salactose linked through
a sulphate ester bond (Lehninger, 1975).
As the cells
adhere equa.lly wel1 to all of these beads, any specif':i.cit:.- is hip:hly suspect.
'~J.rthermore,
when D'?AS-Bio,::el
(positive charge) and CM-BioRel (ne~ative charge) are
used, the cells adhere equally well
30
both tead types.
They are also inhibited by the presence of calc:Lwn ion.
What this ?robably means is that the cells sre adhering
to the
a~arose
bead itself, and the charge produced by
the D:::AF snd CM is negLtc;ible in th:2_s adhesion.
Since this cell-bead adhesion is unlikely to be specific, what then is causing the adhesion?
The evidence
seems to point toward a cherge-eharge, hydros:-r,en bonding,
or van der Waals interaction.
Cnlci1u~
ions and magnes-
hun ions hPve a d-ivalent positive char·ge.
ions inhibit adhesion.
'l'hese nositive
Galacturonic acid and glucuronic
o.cid tend to incres.E>e f,dh:;sion,
V•:hen the CI':1;;'-S 1ir
'i'lEU3
buf-
:[' e:r•c-cl to p::-r 8. 3, botlJ the ga lac t1:ron::tc and g luc:uronic
acid were negatively charge, while the cells had a net
negative charGe•
Therefore, deoending on what charse
the beads hold, with a
sim~le
cha~Ge-charge
interaction,
two d:iff(c;r.snt situations ct= n occur:
I.
Ii' the beads
8Y'e
nos::tively charged:
A) a simple positive-ne3ative attraction, resulting
'
in adhesion
in CMF-SW:
(+J
16
B)
in the presence of calcium ions, inhibition
will occur:
(+)
l+)
·--/~+bead
/
)
/.-
·····" I
the cn lc ium ions surround the cell a.nc1
lize the negative
el:.B.r~·;e
neutra~
found there, thus inter-
fering with the adhesion to the positive bead.
II.
I~'
th.e beads are
ne~;atively
A)
in CMF-SW the cells and
l-l-)
ch&rged:
~eads
would not adhere:
B) . with calc::um ion present, the cells would adhere
to the beads through a HCalcium bridge":
c
(_~-)
t-+
(l-)
0
C) addition of a nesatively c~a~~eJ acid su~ar would
result in
de~reased
(+)
adhesion:
E,cidsugar
{+)
acidsuger
'l'hus, Dccording to the above scheme, if a chE:r'gech2rge interaction is involved, then the cells and beads
will bebsve
charged.
2.s
obser·ved if the beads are PC?siti.vely
They will adhere in an ooposite manner if the
beads are negative.
The results from the bead electro-
18
phoresis show that the Sepharose beads tend to have a net
char~·~e
negative
(see results).
Clearly, a chE·rge-charge
interact ion is 1m likely.
Slectrostatic rorces other than a charge-charge interuction can exist between eells and beads.
bondinc~
results from a hydror:;en atom serv1ng as a
between two electronegative atoms.
"'tC'l''"S·
C....
..} l.l
>.
,"-
Hydrogen
nc-.n o'er" 1''.,olQ
V C".l..
{
'
'-
C. C•.
"-'
~nteroctl'ons
_L
.., 'J C•,
brid£~e
Between non-nolar
PX~
~ J..
0
c-+·
~ U
~
exner:Lmental evidence r;c.int to these types of. interaction
bet1veen the beads and the cells: sodiu"vn J:kriodate treEt-
mont, and the acid sugar and divalent cation results.
Sod~_unl
per•ioda te oxidizes mono- and
polysaccharide;~
resulting in the cleavage of adjacent carbon-carbon bonds.
When cells are treated with periodate, the Polysaccharides
:found :in tbe cell surrace glycoGalyx would be removed ..
The treatment of the sea
urc~·lin
cells v:ith periodr;_te re-
sulted in a reduced adhesion to the bes.ds.
rrhe bead sur-
f.'ac e contains carboxy lie ,:,roups as impurity s, v1hich can
interPct to form
hydro~en
(Porath, 1971).
Since removlng P&r>t of the glycocalyx
bonds with other comnounds
resulted in reduced adhesion, it is entirely possible
tho_t; the
su~~:&r
g:''Oups removed had been 5.nteracting with
the c&rboxylic groups on the beads.
The periodate treat-
ment could have also removed groups on the bead surface
which con':ributed H- to a hydrogen bond between the cells
and the beads.
At pH 8.2, any carboxylic g:roup "''ill be close to
lOOfb dissociated, and will not posses a H- v;hich can bo
used in lzydrogen bondinc.
negativelv charced.
The carboxylic group will be
It is possible that on the cell s1n"-
face there exists compounds which can contribute a H- to
hydrogen bondinr:;, and thus the interaction between the
cell s.nd cr-.rboxylic inmuri ties can still exist.
The Seoharose 4R
un i t
.3 , _ e. n cJ
~ead
is
com~osed
of polygalactose
t h ;J s e c D. n :tnt e r 2 c t vli t [- t h e c e 11 surf a c e t o
produce hydrogen bonding:
if:.:-herc
y
•
""
lS
an electroneg-
ative atom ( 0 or N
In addition to polai' ''roups, there ac:'e present on
the cell surface non-polar grouns v:hich ce.n interc,,-t to
produce van c3er V.'2,als interact"ons.
Vander V.'aals for,ces
are the result o{ an induced dioole between adjacent atons.
\Vhile of ''ery short range, van der Y.'aals are strong
encuc:_:h to hold cells 0ogether:
non~:)olar
phos"holi:Jids
form van der Wsals interactions, and hold the C3ll wall
together.
Both
hydros~m
r·onding f'nc van cler V:ia.sls inter-
actions can be acting tocether to
~roduce
the observed
cell-bead adhesion.
Glucuronic Bcid and galDcturonic acid are
acids, and are nesatively charged at pH 8.2.
either of these compounds to the
cell-~
c;:~rboxylic
Adding
ead st__;spension re-
20
sulted in increased adhesion.
Adding cnlcium or mEgnt}simn
to the cell-bead susnension inhibited
adhesion~
posi~ive
ncsium end c·lciLUn have a divalent
The mag-
cherge.~
These oppositely charged compounds (acid sugars and divalent cations), when add ld to the cell-bead suspension,
acted in an opposite manner.
This tends to indic8te an
el ec tr-ost8. tic in t>n•ac tion (hydro :en bond ins; or van d er
Waals) between th.e cells and the beads.,
How th3 divale.c1t
C8.tions D.nd char[IJ'd sug"":J'S are interr.cting vrith the cells
and beads is unknown.
It thus seems c)ossible that the cells and beads b:Lnd
through a hyd:r>or;en bonding or vE: n d er Via a ls interact ion.
This leaves two unanswered questions: l) what is the signifigance of this interaction in terms of csll-cell adhesion?, and 2) wty does manganese ion not inhibit the
interaction in the same way as calcium,or to a lesser
extent, rrragnesium ion?
I.
The manganese ion
&ction is p1..1.zzling.
ef~ect
upon the cell-bead inter-
Since calcium inhibits cell-bead ad-
hesion so strongly, manganese would be expected to have
a similar 0ffect.
Yet, it seems to increase adhesion, or
at least, not inhibit it at nll.
Eumnhreys (1963) showed
that snonge factor-mediated a ':gregation was specific for
calcium nnd
valent
mar~:nes
cat~ons.
ium, but would not occur wi tb other d i-
Eo7ever, since the
cell-b~ad
interaction
is non-specific, manganese could not be acting in a spe-
21
eific manner.
Manganese is present only as a trace ele-
ment in 1-f:F'-s·w.
'rhe amcnmts that vere acl6::;d in this work
may have :i.ndueed an Brtifaet into the experiment; resulting in increased cell leakase.
To answer the first question, when dissociated cells
come into initial contact, non-specific interactions
could be the first step in adhesion@
inte~actions,
such
88
hydrogen
These non-specific
bondin~
and van der Baals
forces, could be the first int1raction betweon cells in
close contact ..
cells close
'l1 hus,
enou~h
could occur.
those interElJ;tions would druw the
together so that more specific
bindin~
It is possible ttat non-specific binding,
particularly with work using inert substances to which
the eells v'lilJ b:Lnd, co1.J.ld ·)e mist<::ken for someth1.ng
more sirrnifigant.
in beads such as
Interactions with irnnurities present
Senha~ose
shoud always be csrefully
ruled out.
This
~ork
renresents initial attemots at understAnd-
lnr:; cell-bead intersction in a specific system.
on the nature of cell adhesion to
bea~s
Stud:tes
may serve as mod-
to help elucidate the forces involvHd in cell-cell
and cell-matrix adhesion in vivo.
ChipawJky, Stefan, Y.C. Lee, and Saul Ros~~an (1973).
Adh8sion of Cultured Fibroblasts to Insoluble Analogs of
Cell-Surface Carbohydratese
P:eoceedings c.f the Kitional
/:..ea6mriy of Scienct~, USA 7, 2309-2~)12.
Hu:rnpb.r·ey s, T::Jm ( 1963) o
Chemica 1 Di ss o lu tion and in Vitro
Roconstru.etion of Sucnf;e Cell Adhesions 1. Isolati~snd
?1)~r1et.ior!. Derflc)n~itratiorl o.f tlJ.e Conrpol1erJ:ts In·volved.
l)evelonffiental Biology B, 27-47.
Jacobson, 3ruce S., Jon Cronin, and Daniel Ernnton ( 19'1'8).
Counling Polyly2ine td Glass Beads for Plasma M~rnbrane
Is o lat. 5 on.
Bi oc 1:-,iril:i.ca et Biopb: s ice, Acta 5C6, 81-96 ..
LebninP~:.:c,
Albert L. (H175).
'~~oche10j~!Ey, 2nd edition.
Worth Publishers, X.Y., 268.
Che~istry of D-glucuronic Acid and its
Glycosides e
In '~lT,c::J.ron:Lc /\c id, Pree and Ccmb:Ln·3d, T~d ~
by Geof"frcy J-. DutTori·;-·Aca~o enuc· Press~ -r.r:'! ; ;-3-]~19 ..
Varsh, C.A. (1966).
0
~azia,
Daniel, G. ?chatten, and We Sale (1975).
of rells to Surfaces Coated with Polylysinc.
of Cell Biolo~y 66, 198-200~
Adhesion
Journal
Morrison, Fobert Thornton, and Robert Nielson Boyd (1974).
Crr~ar:ic Chemistrv, ?rd edition, Allyn c..nd Bacon, Inc.,
,-cf,"t;·-·-·--·~----~--J/...
__ .l.G..J •
Opponheir,;er, S.R., (1975).
C.ue.ntita.tive Assay for Measuring Cell ~1\.gglutinstion in Small Volumes.
In Cor,canavfi.ll.n A as a 'l'ool, -cc1. by H. Bittiger and ~...r .P. ::Jchneb-ii,
~fciT-iri-··vrrro·y and-Sons, London, 293-299.
Oppenheimer, Steven B., Richard L. Potter, and ~ary Lee
}3,-::;:r_•ber (H1'73).
Alter·ation of ~~ca Urct.in ·~mbryo Cell
Surface ~~roper-ties by :-.r.yeoE;tEd~:ln, 8. Sterol Binding Ar;tibiotic.
Developmental 2ioloby 33, 218-223.
Porath, J e~ker, Jan-Chris ter Janson and Tor E;hy La as ( 19'71) •
Agar De:r> 1 ;_rs_ t i v c s fop Chro1ns. top' a l'f'Y, ,-.1 ec t;ron horcsi s,
and GeJ.-Bonnd }-~nzymes:
I. Desulphatecl and Reduced Cross
linked A~ar and ARarose in Soherical 3ead Form.
Journal
of Ch.romotoeranhy · 60, 167~ 1?"7.
Roseman, Satl1 ( 1970). The f:yn.thes is of Comnlex Carbo:r~y­
drates by li!ll.i.ltiglycosyltransfere.se Systems and The1r
Potential F't.mction in Intercellular Ad>-esion.
and PL_ysic:s of' Lipids 5, 270-29'7.
Chemistry
Rosen, Steven D., John A. Ksfka, David L. Simpson, and
So.muel ,J Barondss (19?3).
Developmentally :Pepuh1ted
Carbohvdrnte-bindinrr Prote5_n in DictyosteLiu.:m cUscoi&
C>U.:ffi 4
d
.._ ..,_,.
•·
;_)-,-,Q (;
.;.
.J.
C>
..:._,..
ed' _, LL
~ ~:
r" O
'o~ ..1r>
....;
_;_
USA 70, 2554-2557.
tJ.-•l J. e
1'_ T'l (_:_:..
Q
i-,:
~-- .J~
on-a
1----}r,-'-('-;;-0~-C>vn;;-·-o·r-~
('
.. <.~.
......_•.~-
._-
....
•,
~ 8-1:::-(-: C>
'~'
· ..1...
J.
,
,
24.
.Pigure 1
Diagr'am of the device used to determine the chG.Y'f'/3
on tb.e beads.
'I'::1e chambers ·;;.rere filled ·with an
elec~~:rcdn
buffer containing 151 su~rose (wt/wt}, and an electric
field was passed across the main chamber.
As the beads
fell through the electrode buffer, their path was traced,
and the surface charge deter:-nined !'rom their defleetiono
(J)
(\
3
I
I
I
E:
I
=--···--~---·
~·
II
fL
_L___ ~
p ---------(\
3
26
Figure 2
Effect
o~
selected monosaccharides upon relative ad-
hesion of cells to Seoharose beads.
cells Dlus beads in CI•E"-EWJ.
In this graph, 0~ is
Points above this line incH-
oate increased adhesion, and below the line indicates decreased adhesion.
Each su8ar was tested in three differ-
ent experiments, and the vertical bars represent the
standard deviation from a pool of all three experiments.
The central dot on each line is
t~e
average value.
,._
c'"
ga lEctos e
mannose
xylose
10
I
t
I
t
+
5
%
a
d
h
0
5
n
---
l!
'
!
,oJ
J.
I\
----
I
s
0
i
~-
e
i
glucose
1
I
l
r
I
I
j
I
7
v
I
f
/
v
~
lo-1 1o-2
~
1()-2
1....... o=3--1~o=1-1c',-2
"
....,..,
--~
•
mo 1. . . es
concentration of sugar ln
lo-3
28
Figure 3
Effect of galacturonic
a~id
Sepharose-cell relative adhesion.
and glucuronic acid upon
These
gars increased the adhesion to the beads.
char~ed
acid su-
Standard dev-
lation indicated by vertical lines, from a pool of three
separate experiments.
29
70
I
60
,,
+
50
crt
/-0
a
d
h
<1()
1
e
s
i
;)Q
0
n
f:O
10
0
"--------·----------------------------------------~
galacturonic acid
glucuronic Ecid
10
concentration in moles
::;o
r ,
"!<'igure 4
Effect of divalent cations uPon relative adhesion of
cells to Sepharose beads.
The strong inhioitor;y
of calc i'lJXIl can be s sen in this f igur· e; as can
latory effect of manganese ion.
thE~
e~'fect
s timu ...
Standard deviation for
a Pool of three experiments shown by the vertical lines.
Increased adhesion-above O o. l_-,
belo-w O% line.
1l• nP
-
•
~''
decr~ased
adhesion-
31
50 -
1------manganese
0
n 10
magnesium
15-
20
25[
30
35
40
r·
t-
t
I
45
50-
f
~~:
'75
l0-5
concentration in moles
:'12
. PJ.gure 5
Effect of 10- 2 M calcium chloride upon previously·
forMed cell-bead aggregations.
The cell-bead clumps would
bec;:i.. n to fall apart upon adc1 it ion of the calc i
CMF-31!! in v1hich the c lumns were suspenc3.ed.
1,:m
to the
25
20
+"
15
10
5
'
0
5
10
15
20
25
---·-·-·-------------t----------- !f-----..1------·
1
2
3
time in minutes
30
34
F:igure 6
Relative 2dhesion of cells to bAads other then Seoharose 4B.
The cells adhered to the beads in
wer·e :tnh:'Lbj_ted by L1F'-2.1!l or calcium ion.
CM~-~v
and
Values ebov e the
O% line indicate increased adhesion, below the line, docreased adhesion.
2oli
+
15
101
a
--~-
d
h
s
t
0
10
n
1.5
20
60
651
70r
75t
80~------------------L------------------~--~~
Bioe;el
P-100
Sephs.dex
Ul tro-
gel
D!::AE
gel
CM
gel·
36
l'' :Lgnr e ?
Apr•etn•ance o:;.'
floating in
C0?-~W.
cell~be~3d
clt.:anns as initially seen
This is a typical view, although with
additional rotation on a shaker, the
compc-,ct.
a~gregation
MagnifieD tion approximately 4X.
will
37
Figure 8
Light
the photo,
micro~-;co':Je
~
to the beads.
view of cell-bead a.0::gregates.
In
is a bead, and £ is a group of cells adhered
Shaking the vial in which the beads were
suspended did not break up the a
~grer<:ates.
240X.
39
<l()
Figure 9
Shan~ing
gates.
electron microgrnoh of the
cel~-bead
aggre-
In this view, ~is a Sephaross 4B bead, and cells
r\;;:,.I
c·' c<·-cl. n be seen
clin~ing
to its surface.
a lar~e number of beads together.
The cells hold
~agnification-864X.
41
--
F'igure 10
A view of the cells e.nd beads as they a DP ear in MF-
SWo
Shaking the vial gently showed that the cells and
beads were not adhered to each other.
In this view,
is a Sepharose 4B bead, and c is a cell.
240Xe
~
~agnification-
43
-I
Figure 11
Photograph of
ses were run.
de~tained
gel on which the glycoside-
The bands in the majority of casas were
. ~t
very 1 1311·,
ana. d o not show up well in this photograph.
A:
~-glucosidase
G-500~
B: «-L-fucosidase F-7753
C: B-R-acetylglucosaminidase A-3015
D: •><-galactos:i.dase G-900'7 (reported by Sig,rna to have less
than 0.5% B-N-acetyle;lucoss.minids.se and B-xylosidase~)
E:
~-mannosidase
M-7257
F: B-galactosidase grade VI, G-6008, reported by Sigma to
contain less than 0.011 contamination (unsPecified).
The bar' ;·oints to a single band found :i.n the B-galactosi-·
dase section, which is barely v:i.sible.
D also contained
a single band, which is not apnarent in this view.
45
4[)
Figure 12
of a pE grsdient unon the reletivs adhesion of
Ef~ect
s0a
urchin cells to Sspharose beads.
The cells
Lrrum ndhesi.on at pH 6.0, normal adhesion at p1J 8
anJ slichtly
hl~her
a~hesion
at nH 10.
~how
~u:td
a min9,
The increased ad-
hesion at pH 5 and 4 is probably due to cell demaze and
leakage of
intercel~ular
material.
4?
+
a
c1
e
s
:t
0
n
~----~-
8
pH
9
10
. Figure 13
Effect of pH upon the relative arlhesion of cells to
DE!-~.':-Biogol.
decrease
~as
I~in1mum
adhEosion anpeared at nE BoO, and a
also seen at pH 5.0.
indicated by the vertical lines.
Standard deviation
i
15
)
I
~10
I
I
't
d
,)
/:i
a
1
d
h
8
s
0
:t
0
n
5
\
10
15 T
I
L----~-----,3
4
____,5_.
6
pH
?
8
9
10