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Journal of Cell Science 101, 873-883 (1992)Printed in Great
Britain © The Company of Biologists Limited 1992
873
Initiation of HeLa cell adhesion to collagen is dependent upon
collagen
receptor upregulation, segregation to the basal plasma
membrane,
clustering and binding to the cytoskeleton
MICHAEL L. LU*, RICHARD J. McCARRON and BRUCE S. JACOBSONt
Department of Biochemistry and the Graduate Program in Molecular
and Cellular Biology, University of Massachusetts, Amherst,MA
01003, USA
•Present address: Brigham and Women's Hospital, Harvard Medical
School, 75 Francis St., Boston, MA 02115, USAtAuthor for
correspondence
Summary
It was recently reported that HeLa cells have three
Arg-Gly-Asp-dependent collagen receptors that do notappear to be in
the integrin family of extracellularmatrix receptors and bind to
either type I or IV collagenor to type I gelatin. It was our goal
to determine howthese receptors function in HeLa cell-substratum
ad-hesion. We report here that the sequence of events bywhich the
receptors mediate adhesion to collagen orgelatin is: (1) induction
of cell attachment by specificcollagen receptor-substratum
interactions with culturedishes covalently coated with either type
I collagen orgelatin - attachment is inhibited by soluble
gelatin;(2) stabilization of attachment by exocytotic upregula-tion
of the receptors to the basal plasma membrane,which was
demonstrated by analyzing, during celladhesion, the redistribution
of the collagen receptorsamong the apical plasma membrane exposed
to theculture medium, the basal plasma membrane contactingthe
culture dish, and an intracellular pool of plasmamembrane vesicles;
(3) the initiation of cell spreading by
receptor clustering and cytoskeletal association. Cellspreading
is a threshold effect with regard to the surfaceconcentration of
gelatin, indicating that collagen recep-tor clustering is a
precondition to the onset of spreading.Observations consistent with
this interpretation of thethreshold effect are that cells attach
but spread moreslowly on a substratum that retards receptor
clustering,and that collagen receptors, when viewed by
immunoflu-orescence microscopy, form a punctate pattern
offluorescence in the basal plasma membrane during cellspreading.
It is also shown that more collagen receptorsco-isolate with
nondenaturing detergent-stable cytoskel-etal preparations after the
collagen receptors have beeneither clustered by antibodies or
gelatin in solution, orby a collagen matrix. This indicates that
clusteringdrives the receptors to bind to the cytoskeleton and is
anecessary step in the transition from cell attachment tocell
spreading.
Key words: cell adhesion, collagen receptors, HeLa cells.
Introduction
Adhesion of cells to an extracellular matrix (ECM) isintimately
involved in cell migration, metastasis andtissue development (e.g.
see Burridge et al., 1988; Buckand Horwitz, 1987; Juliano, 1987;
Thompson-Pletscher,1986). An in vitro model of these normal and
aberrantfunctions is the adhesion of cells to culture dishescoated
with extracellular matrix components. Thegeneral events that are
thought to occur duringadhesion of cells in vitro are: first, an
initial attachmentphase; second, an intermediate phase where the
cellsspread or migrate by forming adhesion zones betweenthe
substratum and the cell; and third, a final phasewhere the cells
either detach or the cell-substratumadhesion zones become more
elaborate or are re-modeled by incorporating existing or newly
synthesized
materials (cf. LeBaron et al., 1988; Singer et al., 1987,1988;
and reviews by Buck and Horwitz, 1987; Juliano,1987; Burridge et
al., 1988; Lark et al., 1985; Geiger,1983; Geiger et al., 1984;
Rollins et al., 1982; Culp etal., 1986; Rees et al., 1977).
Our aim has been to focus on the function of HeLacell collagen
receptors in the first two phases of celladhesion, i.e. cell
attachment and cell spreading. Wehave previously shown that HeLa
cells have three Arg-Gly-Asp-dependent receptors for collagen that
mediatecell-substratum adhesion but do not appear to be in
theintegrin family of ECM receptors (Lu et al., 1989;Beacham and
Jacobson, 1990). The receptors aredesignated by their molecular
mass and that of anyprevalent proteolytic fragments (102/58 kDa, 87
kDaand 38/33 kDa). These cells are less complex than mostother
cells used to study cell adhesion in that they do
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874 M. L. Lu et al.
not make an extracellular matrix, which is thought to beinvolved
in the formation of the more complexadhesion zones referred to as
focal contacts andfibronexuses (Izzard and Lochner, 1976; Singer,
1982;Singer et al., 1987; Couchman et al., 1983; Fairman
andJacobson, 1983; LeBaron et al., 1988). This is animportant
aspect since it is difficult to separate, in time,the formation of
the complex adhesion zones from theattachment and spreading events.
Therefore, sinceHeLa cells do not form complex adhesion zones, we
areable to dissect the sequence of events that take placeduring
attachment and spreading.
The objectives of the work reported here were todetermine (1)
whether the HeLa cell collagen receptorsact cooperatively to
mediate firm cell attachment; (2)whether the collagen receptors
segregate to the basalplasma membrane (PM) by diffusion from the
apicalPM and/or are upregulated by exocytotic membraneflow from an
internal pool of PM vesicles; (3) whetherHeLa cell spreading on
collagen is a cooperativeprocess, and whether such cooperativity is
due toclustering of the collagen receptors; and (4)
whetherclustering collagen receptors induces them to associatewith
the cytoskeleton to initiate cell spreading.
Materials and methods
Cell cultureSuspension cultures of HeLa-S3 (ATCC) cells were
grown tomid-log phase (3-5 x 10s cells/ml) at 37°C in a humidified
5%CO2 incubator in RPM 1640 medium (K.C. Biologicals,Lenexa, KS)
supplemented with 5% calf serum (Gibco,Grand Island, NY), 0.3%
NaHCO3, 100 /ig/ml dihydrostrep-tomycin, 60 /
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HeLa cell-substratum adhesion 875
at 970 g yielding a pellet containing the silica-coated
apicalplasma membrane fragments and a supernatant containingthe
cytoplasmic fraction. The cytoplasmic fraction was thencentrifuged
with a microfuge at 14,500 g for 15 min to obtain afraction
containing large intracellular vesicles termed "theinternal plasma
membrane." The basal plasma membraneattached to the culture dish
was washed two times with coldPBS and scraped into 2% SDS
containing the proteaseinhibitor cocktail. The "internal PM"
fraction was separatedfrom the cell surface PM the same way as that
done with theapical PM and the internal membrane fraction, except
theentire cell surface for the cells in suspension was coated
withcolloidal silica and the cells were disrupted in a parr
pressurebomb at 600 psi. All fractions were solubilized in the
sameSDS-protease inhibitor cocktail. The samples were heated to90°C
for 10 min and sonicated with a Branson sonifier(Branson
Ultrasonics Co., Danbury, CT) set at 35 W andsonicated 3 times for
5 s durations. The microbeads and otherinsoluble materials were
then removed by microfuging thesamples for 5 min.
Indirect immunofluorescence microscopyHeLa cells in suspension
or after different times of spreadingon gelatin-coated culture
dishes were fixed with 3% parafor-maldehyde in PBS, pH 7.4, for 30
min on ice. Cells weregently washed three times in PBS then
incubated on ice for 5min in 0.1 M glycine in PBS, pH 7.4, to block
residualformaldehyde groups. After one wash with PBS, the cellswere
incubated on ice for 30 min in PBS containing 1% BSAand mouse
primary antibodies to the collagen receptors. Cellswere then washed
with PBS containing 1% BSA anddecorated with FITC-conjugated rabbit
anti-mouse secondaryantibodies for 30 min on ice, after which the
cells were washedextensively with PBS/BSA. The specimen samples
werecovered by one drop of 3% (w/v) rc-propylgallate in 90%
(v/v)glycerol.
Gel electrophoresis and electroblottingSDS-polyacrylamide gel
electrophoresis (SDS-PAGE) wasperformed using a 4% stacking gel and
an 8% resolving gel inthe presence of /J-mercaptomethanol. Proteins
were electro-phoretically transferred from 8% slab gels to
nitrocellulosesheets as described by Towbin et al. (1979). For
staining withantibodies, the nitrocellulose blots were blocked with
150 mlNaCl, 50 mM Tris, pH 7.5, containing 5% non-fat dry
milk(Carnation Co., Los Angeles, CA). The decoration ofprimary
antibodies with alkaline phosphatase-conjugatedsecondary antibody
was completed in the same buffer. Theunbound antibody was removed
by washing extensively inwash buffer (150 mM NaCl, 50 mM Tris, pH
7.5, containing1% Triton X-100, 0.5% sodium deoxycholate and 0.1%
SDS)and the color was developed by incubating in
5-bromo-4-chloro-3-indoyl phosphate p-toluidin salt (Sigma) as
chromo-genic enzyme marker (Leary et al., 1983).
Results
HeLa cell adhesion to collagen-coated culture dishesfollows the
same characteristic spreading morphology(Fig. 1) as that seen with
the adhesion of most cells invitro. Round cells attach to the
substratum and spreadby continually sending out new lamellipodia
andfilopodia at the edges of the cell as the spaces betweenthe old
lamellipodia and filopodia fill in with proto-plasm by a process
called webbing (Rajaraman et al.,
Fig. 1. HeLa cell attachment and spreading on collagen-coated
culture dishes. Scanning electron micrographs, (A,B and C). Light
micrographs of hematoxylin-stained cells,(D, E and F). Cells were
allowed to attach for 1 min (Aand D), and spread for 15 min (B and
E), or 60 min (Cand F). Bars, 5 fjm (A,B), 10 /im (C) and 20 jan
(D, Eand F).
1974; Knox, 1981). HeLa cells which attached to rat tailtype I
collagen or gelatin were partially spread by 15min (Fig. 1, B and
E) and fully spread by 60 min (Fig. 1,C and F). The collagen or
gelatin is covalently bound tothe culture dish since cell
attachment is reduced andattached cells do not spread if the
collagen or gelatin isonly adsorbed to the dishes. Experiments
showed thatadsorbed 125I-labeled gelatin is released from
theculture dish during cell adhesion (data not shown).Desorption or
removal of fibronectin from focaladhesion sites during fibroblast
spreading has also beendemonstrated (Avnur and Geiger, 1981;
Grinnell,1986). In addition, adsorbed fibronectin is evenremoved by
antibodies to the surfaces of cells used inspreading assays to
determine whether a particularantigen is involved in cell adhesion
(Chen et al., 1985).For these reasons all experiments on cell
attachmentand spreading reported here were done with
covalentlybound substrata.
Specificity of cell substratum adhesion to variousphysical forms
of collagenThe rate of HeLa cell attachment (Fig. 2) and
spreading(Fig. 3) on gelatin or collagen is rapid and the
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876 M. L. Lu et al.
100 -
Fig. 2. Kinetics of HeLa cell attachment to culture dishescoated
with gelatin or a positively charged polymer. HeLacells were
metabolically labeled with [3H]thymidine for 48h, suspended in
HEPES-buffered RPMI-1640 (seeMaterials and methods) and plated onto
gelatin-coatedculture dishes at 37°C (open squares) or 20°C
(opencircles), or at 37°C onto culture dishes coated
withpolyethyleneimine (open triangles). Attached cells
weresolubilized in 0.1 M NaOH and quantitated in a
liquidscintillation counter. The vertical bars are the
standarderror of the mean for four separate determinations.
percentage of attached cells that spread is high.Generally,
85-95% of the attached cells spread (Fig. 3;cf. Mason and Jacobson,
1985). While attachment israpid on a positively charged substratum
such aspolyethylenimine (PEI), cell spreading is
significantlyslowed (Figs 2 and 3). Low temperature (20°C)
alsosignificantly reduced cell attachment (Fig. 2) andinhibited
cell spreading (data not shown). Interestingly,while cells attached
and spread at 37°C on culture dishescovalently coated with gelatin
at room temperature,cells attached but did not spread at 37°C on
culturedishes covalently coated with gelatin at 45°C (Fig. 3). Itis
possible that the gelatin bound to the culture dishes at45°C
further denatures its cell binding conformation aswould be expected
of gelatin coated on dishes at roomtemperature. Gelatin molecules
above their criticalmelting temperature of 41°C are in an
extendedconformation and do not have a tertiary structure asdoes
gelatin below its critical melting temperature.Below 41°C even
gelatin has alpha helices, although thehelix is not the same as the
quaternary, triple-helicalstructure of collagen (cf. Traub and
Piez, 1971).
Cell attachment and spreading on gelatin are thresholdphenomenaA
convenient way to ascertain if a receptor-ligandinteraction might
be a cooperative reaction is todetermine whether it has a threshold
or criticalconcentration below which the reaction does not take
8 0
Fig. 3. Kinetics of HeLa cell spreading at 37°C on culturedishes
covalently coated at either 22°C with gelatin (opensquares) or
polyethyleneimine (open triangles), or at 45°Cwith gelatin (open
circles). Cells in HEPES-bufferedRPMI-1640 were plated onto all
culture dishes at 37°C (seeMaterials and methods). At the times
indicated thepercentage of attached cells that spread was
determinedusing a phase contrast inverted Nikon microscope.
Thevertical bars are the standard error of the mean for
fourseparate determinations.
place (Weigel et al., 1978, 1979; Aplin and Hughes,1981; Oka and
Weigel, 1986). We followed this kineticapproach with HeLa cell
attachment and spreading ongelatin. Gelatin was used instead of
collagen since (1)HeLa cells attach and spread equally well on
collagen orgelatin (Figs 1, 2 and 3); (2) HeLa cell
collagenreceptors bind to both type I collagen and gelatin, andtype
IV collagen; (3) antibodies to the collagenreceptors inhibit cell
spreading on gelatin or conversely,substitute for gelatin and
facilitate cell spreading (Lu etal., 1989); and (4) it is easier to
control the amount anddistribution of gelatin bound to the
sulfonated poly-styrene culture dishes. Collagen aggregates in
solutionsused to covalently couple it to the culture
dishesresulting in an uneven surface distribution.
Cell attachment was followed by metabolicallylabeling the cells
with [3H]thymidine. The percentageof cells attached was determined
from the percent oftrichloroacetic acid-precipitable counts
associated withthe culture dish. The percentage of cells attached
todishes with different surface concentrations of gelatindiffers
depending upon the time of incubation beforethe unbound or weakly
attached cells are washed away.If the percentage of cells attached
is determined after 2or 4 min incubation and plotted against the
gelatinconcentration on the dish, a sigmoidal relationship
isobserved (Fig. 4). After 2 min incubation, the concen-tration of
gelatin at which cells begin to attach isbetween 18 and 20 ^g/cm2,
clearly indicating a threshold
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HeLa cell-substratum adhesion 877
CO
.28
3?
100-
10 20 0Gelatin (/jg/cm2)
10 20
Fig. 4. HeLa cell attachment at various times of incubationas a
function of the amount of gelatin on the culture dish.HeLa cells
were metabolically labeled with [3H]thymidinethen suspended in
HEPES-buffered RPMI-1640 containing1 mg/ml BSA, plated onto culture
dishes with differentconcentrations of gelatin and incubated at
37°C (seeMaterials and methods) for either 2, 4, 10 or 20 min
asindicated in the upper left corner of the figure panels.
Cellswere incubated in either the absence (open circles) or
thepresence (filled triangles) of 1 mg/ml gelatin in theincubation
medium. The vertical bars are the standarderror of the mean for
four separate determinations.
effect. The sigmoidal relationship disappears withlonger times
of incubation (Fig. 4, 10 and 20 min).Additionally, gelatin in
solution at 1 mg/ml blocks theattachment, which is consistent with
the reasoning thatspecific cell surface receptors are involved in
theattachment (Lu et al., 1989).
The percentage of attached HeLa cells that spread in60 min was
also found to have a threshold concentrationof gelatin below which
the cells do not spread. Theconcentration is 19 to 20 jUg/cm2 of
culture dish surface(Fig. 5). Interestingly, when percent cell
spreading wasmeasured after 15 min, when the cells were
onlypartially spread, the threshold concentration of gelatinwas
still 19 to 20 jig/cm2 (data not shown). In otherwords, the cells
below the threshold concentration donot even partially spread.
Segregation of collagen receptors to the basal PMduring cell
spreadingThe distribution of the receptors in HeLa cells in
3
-
878 M. L. Lu et al.
son, we also probed the blots with antibodies to a 45kDa
collagen-binding protein (45 kDa CBP) notthought to be involved in
HeLa cell adhesion to gelatin(Lu et al., 1989). The relative
abundance of thereceptors in the immunoblots was quantified with
avideo densitometer and the results are expressed as the
Fig. 6. Immunofluorescence staining of HeLa cells withantibodies
raised against collagen receptors (CR) orcollagen binding proteins
(CBP). HeLa cells were fixed in3% paraformaldehyde and stained with
either anti-102/58kDa CR in suspension (A) or after spreading 60
min (B),or with anti-45 kDa CBP after 60 min of spreading
(seeMaterials and methods) (C). FITC-conjugated goat anti-mouse IgG
was used for visualization with a Dialux 20Leitz fluorescence
microscope. Bar, 20 fim.
percent of total for each receptor in the PM fractions. Inthe
case of cells in suspension, the percent of total for areceptor is
given only for the internal PM fraction withthe balance being in
the external or cell surface fraction.For cells spreading, the
percent of total is given for allthree PM fractions, i.e. the
internal PM domain, andthe apical and basal PM domains that make up
the cellsurface. The most obvious finding is a decrease inrelative
abundance of all the receptors in the internalPM fraction during
cell adhesion (Fig. 7, bars desig-nated INTERNAL). For example, 45%
of the total102/58 kDa collagen receptor is found in the internalPM
fraction when cells are in suspension (open bar)while only 27% for
cells spread for 15 min (diagonallyhatched bar) and 22% for cells
spread for 60 min(horizontally hatched bar). All the collagen
receptorsincreased in the basal PM fraction during cell
spreading(Fig. 7, bars designated BASAL) and tended todecrease or
remain the same in the apical PM fraction(Fig. 7, bars designated
APICAL). In all cases therelative abundance of the collagen
receptors wasgreatest in the basal PM, which accounts for 27% of
thetotal PM in cells spread for 60 min (cf. Mason andJacobson,
1985; Mason et al., 1987).
Association of collagen receptors with the cytoskeletonIt has
been hypothesized that cell attachment andspreading might be
mediated by the segregation ofgelatin receptors to the basal plasma
membrane domainwhere they become clustered and bind to the
cytoskel-eton (cf. Rees et al., 1977; Grinnell, 1978, 1980; Rubinet
al., 1984; Geiger, 1983; Cody and Wicha, 1986;Rapraeger et al.,
1986; Jacobson, 1988). To determinewhether clustered HeLa cell
collagen receptors bind tothe cytoskeleton, cytoskeletal
preparations with associ-ated PM proteins were made by extracting
the cells with
70
60
50
• In suspension 102/58 kDa CRE3 spread 15 minB spread 60 min
40
30
20
100
70
60
50
40
30
20
10
0
Fig. 7. Redistribution of collagen receptorsamong the apical PM,
the basal PM and aninternal pool of PM vesicles during HeLa
celladhesion on gelatin. PM domains wereisolated from HeLa cells in
HEPES-bufferedRPMI-1640 in suspension or after 15 and 60min of
spreading on gelatin-coated culturedishes, and the relative amounts
of threecollagen receptors designated 102/58 kDa CR,87 kDa CR and
38/33 kDa CR, and one 45kDa collagen-binding protein (CBP) in
eachof the PM fractions was determined byimmunoblotting (see
Materials and methods).The internal PM domain, the apical PMdomain
and the basal PM domain aredesignated, respectively,
INTERNAL,APICAL and BASAL under the series ofbars for cells in
suspension (open bars), cellsspread 15 min (diagonally hatched
bars) orcells spread 60 min (horizontally hatchedbars). The
relative amount of each receptor isplotted as a percent of the
total. The percentof total CR or CBP for cells in suspension is
given for only the internal PM domain, with the balance being in
the external or cell surface PM. The percent of total forcells
spreading on gelatin is given for the internal PM, apical PM and
basal PM domains. The vertical lines on the bars arethe standard
error of the mean for four separate experiments.
INTERNAL APICAL BASAL INTERNAL
Plasma membrane domains
APICAL BASAL
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HeLa cell-substratum adhesion 879
Table 1. The percent of HeLa cell collagen receptorsassociated
with the Triton X-100 insoluble cytoskeletal
fractionCollagen receptors
Treatment 102/58 kDa 87 kDa 38/33 kDa
ControlAntibodyGelatinSpread 15 minSpread 60 min
47±370±972±271±472±8
45±468±766±572±874±9
8±494±669±591±591±5
The data are an average of five experiments with the
standarderror of the mean. Control, cells in suspension extracted
withdetergent; Antibody, cells extracted after they were incubated
insuspension with antibodies to the collagen receptors; Gelatin,
cellsextracted after they were incubated with 1 mg/ml gelatin;
Spread15 min, cells were allowed to attach and spread on
gelatin-coatedculture dishes for 15 min before extraction with
detergent; andSpread 60 min, cells were allowed to attach and
spread for 60 min.See Materials and methods for conditions of
Triton X-100extraction and for detection of collagen receptors in
the detergent-stable cytoskeletal fraction transferred to cellulose
nitrate sheets.
a nondenaturing detergent (e.g. see Flanagan andKoch, 1978;
Prives et al., 1982; Woods et al., 1986;Cody and Wicha, 1986;
Rapraeger et al., 1986; Pattonet al., 1989). The intent of the
experiments reportedbelow was to determine the baseline level of
collagenreceptor binding to a detergent-resistant
cytoskeletalfraction, and to determine whether larger amounts
ofreceptors co-isolate with the cytoskeletal fractionduring cell
adhesion.
Nondenaturing detergent-resistant cytoskeletonswith associated
collagen receptors were prepared fromHeLa cells: (1) treated in
suspension with polyclonalantibodies raised against collagen
receptors; or (2)treated in suspension with 1 mg/ml gelatin; or (3)
afterthe cells were allowed to partially spread (15 min); or(4)
after the cells were allowed to fully spread (60 min)on
gelatin-coated culture dishes. The cells were thenextracted with 1%
Triton X-100 in PBS on ice for 20min. After the supernatants were
removed, the cellswere extracted once in PBS without the detergent.
Thesupernatants and the insoluble material were solubil-ized in SDS
and resolved by electrophoresis. Therelative amounts of collagen
receptors in either thesupernatant or the cytoskeletal fractions
were deter-mined by immunoblotting using mouse
anti-collagenreceptor antibodies. The relative abundance of
thereceptors in each fraction was determined by video-densitometry
and the percentage in the cytoskeletalfraction is presented in
Table 1. In all cases, incubatingthe cells with polyclonal
anti-receptor antibodies or 1mg/ml gelatin to cluster the
receptors, or allowing thecells to partially spread for 15 min or
fully spread for 60min, increases the amount of receptors
associated withthe cytoskeletal fraction (Table 1). The
correlationbetween the increased receptor-cytoskeletal
associationdue to antibody or gelatin presentation and
cellspreading is consistent with the hypothesis that spread-ing is
initiated by receptor-cytoskeletal binding that
comes about as a consequence of receptor clustering inthe basal
PM.
Discussion
Recently, work in cell attachment has focused on theinteraction
of specific cell surface receptors with specificECM components (cf.
reviews by Ruoslahti et al., 1985;Buck and Horwitz, 1987; Hynes,
1987) but there is alarge body of information indicating that
attachmentcan also be mediated by nonspecific physicochemicalforces
(e.g. see Maroudas, 1975, 1977; Curtis andMcMurray, 1986). HeLa
cell attachment is initiated byboth nonspecific substrata such as
BSA and the specificsubstrata, gelatin or collagen (Figs 1 and 2).
However,the cells do not spread on the nonspecific
substrataalthough they will spread slowly on the positivelycharged
polymer PEI (Fig. 3; Fairman and Jacobson,1983). Other cells have
been shown both to attach andspread on nonspecific substrata, but
unlike HeLa cellsthey are capable of making their own ECM (cf.
Damskyet al., 1984; Lark et al., 1985).
Experiments to measure attachment kinetics (Fig. 4)and
experiments designed to follow the segregation ofreceptors to
different PM domains during cell adhesion(Fig. 7) indicate that
HeLa cell attachment to gelatininvolves the upregulation of
collagen receptors byexocytosis to the basal PM. The kinetic
measurementsindicate that HeLa cell attachment is a threshold
effectwith regard to the surface concentration of gelatin (Fig.4)
and is consistent with previous suggestions (Oka andWeigel, 1986;
Weigel et al., 1978) that a minimumnumber of substratum-receptor
interactions must takeplace before the cells can attach. However,
thereappears to be more to attachment than just a minimumnumber of
initial receptor-substratum interactions,since with increased times
of incubation (10 or 20 min),the threshold effect of attachment is
dampened and thecells become more adherent at lower surface
concen-trations of gelatin (Fig. 4). These results indicate thatthe
initial contacts of the cell with gelatin become morestable over
time. Consistent with this interpretation isthe observation that,
as the cells are allowed to attach tothe culture dish for longer
times, gelatin in solutionbecomes less effective at inhibiting cell
attachment (Fig.4). The increased strength of attachment does
notappear to be simply a result of the onset of spreadingbecause
the decrease in inhibition of cell attachment bygelatin occurs with
dishes coated with surface concen-trations of gelatin either above
or below that whichinduces cell spreading (Fig. 5). The more firm
attach-ment with longer incubation times at surface concen-trations
of gelatin below the threshold for cell spreadingis consistent with
a time-dependent increase in thenumber of collagen receptors in the
basal PM.
Previous work indicates that cell attachment is mademore strong
by a nonspecific substratum attachment-induced exocytosis of
collagen receptors from anintracellular pool of PM. We have shown
that HeLacells in suspension have 55% of their total PM protein
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880 M. L. Lu et al.
in intracellular vesicles as indicated by cell
surfaceiodination. Upon cell attachment to gelatin, whichinduces
cell spreading, or to BSA, which does notinduce spreading, there is
a stimulation of membraneefflux from an intracellular pool of PM,
presumably byexocytosis. During the first 10 min of spreading
theintracellular pool decreases to 35% of the total PM.This is
followed by a re-endocytosis over the next 20min, so that when the
cells are fully spread on gelatinthe internal pool contains 46% of
the total PM. 55% ofthe PM is found in the internal membrane pool
ofunspread cells attached to BSA (Mason et al., 1987).This
information taken together with the results on theredistribution of
collagen receptors during cell adhesion(Fig. 7) indicates that the
major movement of collagenreceptors to the basal PM domain probably
occurs by anonspecific substratum attachment-stimulated
exocy-tosis. It cannot be concluded that the exocytosis ofreceptors
from the intracellular pool of PM vesiclesoccurred only in the
direction of the basal PM and notthe apical PM. HeLa cells are like
many other cells inthat they constantly recycle their plasma
membranebetween an internal domain and the cell surface(Mellman et
al., 1980; Fishman and Cook, 1982;Widnell et al., 1982; Mason et
al., 1987). It is possiblethat the attachment-stimulated
upregulation of collagenreceptors during HeLa cell adhesion
occurred byrandom exocytosis from the internal PM domain toboth the
apical PM and the basal PM.
Kinetics of HeLa cell spreading revealed that there isa
threshold concentration of gelatin on the culture dishbelow which
the cells will not spread (Fig. 5). This canbe interpreted in three
ways with regard to the initiationof cell spreading (cf. Weigel et
al., 1978, 1979):(1) there must be a minimum number of
receptor-gelatin interactions required for spreading to occur;(2)
gelatin on the culture dish must be present at asufficiently high
concentration where it assumes aunique conformation which only then
allows the HeLacells to spread; or (3) the collagen receptors must
beclustered before cell spreading can take place. It is notlikely
that the number of receptors in the basal PM isthe limiting factor
in cell spreading. HeLa cells left incontact with dishes coated
with low levels of gelatin stilldo not spread (Fig. 4) even though
they increase theirstrength of attachment, presumably by delivery
ofreceptors to the basal PM (see above). It is also unlikelythat a
conformational change in the substratum-boundgelatin takes place at
the higher surface concentrationsof gelatin. Transmission electron
microscopy demon-strated that gelatin covalently bound to the
surface ofpolystyrene cell culture microcarriers at
surface-satu-rating levels maintains a random appearance of
over-lapping fibrous molecules and does not form anydetectable
supercoiled structures or regions of higherdensity (Fairman and
Jacobson, 1983). Thus, thecooperativity in cell spreading is
consistent with theneed to cluster receptors.
To further understand whether cooperativity in cellspreading is
indicative of receptor clustering, weemployed immunofluorescence
microscopy using anti-
bodies to the receptors to determine if visible clustersare
formed during cell adhesion. Antibodies to collagenreceptors that
inhibit cell spreading on gelatin-coatedculture dishes and
conversely, substitute for the gelatinon the dish and facilitate
cell spreading (Lu et al., 1989),exhibit a diffuse fluorescence
staining pattern when thecells are in suspension and a punctate
pattern in thebasal PM of spread cells (Fig. 6). On the other
hand,antibodies to a cell surface collagen-binding proteinthat does
not inhibit spreading or substitute for gelatinas the extracellular
matrix (Lu et al., 1989) give adiffuse staining pattern of
fluorescence in both suspen-sion and spread cells (Fig. 6).
Punctate patterns offluorescence for extracellular matrix receptors
thatmediate cell-substratum adhesion have been repeatedlyshown with
fibronectin receptors (cf. Chen et al., 1985;Damsky et al., 1984;
Giancotti et al., 1986), collagenreceptors (Mollenhauer et al.,
1984) and cell surfaceproteoglycans (Rapraeger et al., 1986).
Furthermore,extensive morphological studies indicate that there is
aco-localization of the punctate fluorescence of thereceptors and
cytoskeletal elements (e.g. Rogalski andSinger, 1985; Chen et al.,
1985).
To further explore whether clustering of collagenreceptors is
essential for HeLa cell spreading, wefollowed the adhesion of the
cells to a positivelycharged substrate, polyethyleneimine (PEI).
The rateof spreading was significantly slowed relative to
cellspreading on collagen or gelatin (see Figs 2 and 3).
Therationale was that if receptor clustering is a precon-dition to
cell spreading, then cell spreading will beimpeded on a substrate
that has a high positive charge,which slows the rate of lateral
diffusion and therefore,receptor clustering. Positive substrates
have beenshown to bind so tightly to the plasma membrane thatthe
lateral diffusion of Con A receptors is restrained(Patton et al.,
1990). It is likely the PEI substratenonspecifically binds to the
collagen receptors and notnecessarily at the "active" collagen
binding site. Whileit cannot be ruled out that the receptors might
not be inthe proper conformation to initiate cell spreading,
itshould be noted that monoclonal antibodies that bind toEGF or
insulin receptors, at sites other than thehormone binding sites,
still induce receptor clusteringand endocytosis (cf. Schlessinger
et al., 1983; Maron etal., 1984; Forsayeth et al., 1987).
Furthermore, endo-cytosis of membrane proteins is induced by
binding tohighly cationized ferritin (Simionescu et al., 1981).
Theabove, taken together with the observation that col-lagen
receptors exhibit a punctate pattern of fluorescencein the basal PM
of spread cells, and that cell spreading is athreshold function of
the gelatin concentration on theculture dish, strongly supports the
hypothesis thatreceptor clustering is a requirement for cell
spreading.
The observation that clustering HeLa cell collagenreceptors
causes them to bind to the cytoskeleton asdetermined by
nondenaturing detergent extraction(Table 1), and experiments
indicating that the gelatinsubstratum clusters collagen receptors
(Figs 5 and 6) areconsistent with previous work with other cells
indicatingthat receptor-cytoskeletal binding must occur before
-
HeLa cell-substratum adhesion 881
cell spreading can take place (e.g. see Rees et al.,
1977;Grinnell, 1978; Geiger, 1983). However, the mechan-ism by
which the HeLa cell collagen receptors interactwith the
cytoskeleton is not known. Clustering lamininreceptors (Cody and
Wicha, 1986) or a transmembraneproteoglycan that is an
extracellular matrix receptor(Rapraeger et al., 1986) induce them
to bind to adetergent-resistant cytoskeletal fraction. Both the
lam-inin receptor (Brown et al., 1983) and the
proteoglycanextracellular matrix receptor (Rapraeger and
Bern-field, 1982) bind directly to F-actin in vitro.
Fibronectinreceptors have been shown to bind to talin in
vitro(Horwitz et al., 1986).
It should be emphasized that the measurements
ofreceptor-cytoskeletal binding determined by non-denaturing
detergent extraction procedures do notindicate whether the
receptors that were associatedwith the cytoskeleton in the absence
of an extracellularligand or substratum were either unclustered or
clus-tered. The data only indicate that clustered receptors,or
making larger clusters from smaller ones, arecorrelated with an
enhancement of receptor associationwith the cytoskeletal
preparations (cf. Brandts andJacobson, 1983). If the receptors were
in the form ofdimers, trimers, etc., and exhibited a weak or even
astrong affinity for the cytoskeleton, clustering them intolarger
oligomers would markedly increase the affinity(Brandts and
Jacobson, 1983; Shiozawa et al., 1989).For example, one estimate of
the enhanced affinityreveals that, depending upon the mole fraction
of thereceptors in the plane of the membrane, a tetramericcluster
would bind to the cytoskeleton 1012 times moreeffectively than four
unclustered receptors and anoctomer 1027 times (Jacobson, 1988). It
should benoted, that these increases in cytoskeletal binding
uponreceptor clustering are consistent with the cooperativityseen
in cell spreading versus the concentration ofgelatin on the culture
dish (Fig. 5).
Is the clustering and binding of the collagen receptorsto the
cytoskeleton sufficient to induce cell spreading orare other
messages involved? It is tempting to speculatethat clustering might
induce a second messenger whichsignals the cells to spread. It has
been shown thatmutants of Chinese hamster ovary cells that do
notadhere to fibronectin have an altered type I proteinkinase.
Adhesion of these mutants can be induced bythe addition of cyclic
AMP (Cheung and Juliano, 1985;Cheung et al., 1987). It is also
known that one of thesubunits of chick fibronectin receptor has a
proteinkinase C substrate-binding site in its cytoplasmicdomain
(cf. Burridge et al., 1988). Interestingly,Danilov and Juliano
(1989) recently reported thatphorbol ester, a protein kinase C
activator, does notalter the phosphorylation state of fibronectin
receptoror talin, or the number or affinity of cell
surfacefibronectin receptors, and concluded that the increasein
cell adhesion induced by the phorbol ester is not dueto a direct
effect on the receptors. Work is currently inprogress to determine
whether a second messenger isinvolved in signaling HeLa cell
spreading. Preliminaryexperiments indicate that HeLa cells have a
dramatic
rise in cyclic AMP and a spike of intracellular freecalcium
during cell spreading; however, both the riseand the spike occur
after spreading begins indicatingthat neither cyclic AMP nor Ca are
the secondmessengers initiating cell spreading (Lu, Chun
andJacobson, unpublished observations).
We are deeply indebted to Dr Peter Mason and DeidraGramas for
the micrographs of spreading HeLa cells, and toMr. Jang-Soo Chun
for providing us with his initial obser-vations on second
messengers during cell adhesion. This workwas supported in part by
a grant from the National Institutesof General Medical Sciences, GM
29127.
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(Received 5 November 1991 • Accepted 15 January 1992)