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Cell spreading factor
Printed in Sweden Copyright @ 1976 by Academic Press, Inc. A// rights of reproduction in any form reserved
Experimental
CELL Occurrence
Cell Research 102 (1976) 51-62
SPREADING
FACTOR
and Specificity
of Action
F. GRINNELL Department of Cell Biology, University of Texas, Southwestern Medical School, Dallas, TX 75235, USA
SUMMARY A factor required for spreading of substratum-attached baby hamster kidney cells (BHK), Chinese hamster ovary (CHO) cells, HeLa cells, and L cells has been isolated and purified from fetal calf serum. A similar factor has also been found in calf, porcine, human, rabbit, and chicken sera. The spreading factor was active when adsorbed to the substratum and prior adsorption of other proteins prevented cell spreading, regardless of the addition of spreading factor or unfractionated serum to the incubation medium. Antibody against the fetal calf spreading factor inhibited the spreading activity associated with unfractionated fetal calf serum and also the spreading activity associated with calf serum and porcine serum. In model system studies it was found that antibody against BHK cell surfaces induced cell spreading when the antibody was adsorbed to the substratum; when it was present in the incubation medium as well as on the substratum, cell spreading was not observed. The data are discussed in terms of the hypothesis that there is a specific serum factor which adsorbs to the substratum surface and is thereby activated, and which then forms the target for certain cell surface receptors. Interaction between adsorbed-activated factor and cell surface receptors leads to cell spreading.
The attachment of baby hamster kidney (BHK) cells in serum-containing medium to non-living substrata can best be described as a series of steps: adsorption of serum components onto the substratum, contact between the cells and substratum, initial attachment, and progressive attachments leading to cell spreading and an increased strength of cell attachment. Although there is a close interdependence of these events, especially contact and initial attachment, they have all been experimentally distinguished by biochemical techniques [l-5]. Some time ago, Wolpert et al. [6] reported that the spreading of BHK cells required the presence of serum in the medium. This has also been reported for HeLa cells [7] and L
cells [8], and for the normal morphology of cell spreading of MCR-5 cells [9]. It is likely that in serum-containing medium the substratum becomes coated with serum components and that attachment occurs to the adsorbed serum layer and not directly to the substratum [2, 4, 5, 7, 9, 10-141. Thus, we have become interested in determining whether the ability of cells to spread onto serum-coated substrata is a general property of protein-coated substrate or a specific property dependent upon certain serum components. In preliminary experiments we reported evidence suggesting that there was a requirement for specific serum components in the spreading of BHK cells [5]. We have now identified a specific Exp
Cell
Res
102 (1976)
52
F. Grinnell
Figs l-3,5. For further details, see “Methods”. Fig. 1. Assay of spreading factor. The substrata
centrations of (a) 3.1 pg/ml; (b) 6.2 Kg/ml; and (c) 15.5 pg/ml. X290.
spreading factor in serum and in this paper report on its biological occurrence and specificity.
Materials
trypsin (type XI, 7 300 units/mg), L-1-tosylamide-2uhenvlethvl chloromethvl ketone and phenylmethyl sulfonyl fluoride were purchased from Sigma Chemical Co., St Louis, MO. Whole bovine gamma globulins (fraction II) was a product of Miles Laboratories, Kankakee, Ill. Whatman DE-52, DEAE cellulose, was purchased from Reeve Angel, Clifton, N.J. Bio-Gel A 1.5 m was the product of Bio-Rad Laboratories, Richmond, Calif. Other reagent grade chemicals were obtained from Fisher Scientific, Houston, Texas.
Baby hamster kidney (BHK) cells which were suspension-culture adapted (BHK21-13s) were the gift of Dr Adrian Chappel, Communicable Disease Center, Atlanta, Ga. Polyoma-transformed BHK (BHK-py) cells were the gift of Dr Walter Eckhart, The Salk Institute, San Diego, Calif. Normal BHK (BHK21-C13) cells, NCTC clone 929 (L) cells, and HeLa cells were obtained from the American Type Culture Collection, Rockville, Md. Chinese Hamster Ovary A10 (CHOAlO) cells were the gift of Dr William Garrod, Southwestern Medical School, Dallas, Texas. Eagle’s Minimal Essential Medium (spinner modified), McCoy’s 5a medium (modified), fetal calf, porcine, calf, chicken and rabbit sera, MEM amino acids, MEM vitamins, and trypsin 0.25% were obtained from GIBCo, Grand Island, N.Y. Human serum was purchased from Microbiological Associates, Bethesda, Md. Bovine serum albumin (type F), (ConA), N-ethyl-maleimide,
BHK21-13s and BHK-py cells were grown in suspension culture and BHK-21-Cl3 cells were mown in stationary culture. The culture medium was Eagle’s MEM (spinner modified) with double the concentration of amino acids (except lxglutamine) and vitamins and supplemented with Hepes buffer (20 mM), 0.1 g/l ferric nitrate, 2.0 g/l dextrose, 10% tryptose phosphate broth, and 10% fetal calf serum. The final sodium bicarbonate concentration in the medium was 0.5 p/l. HeLa. CHO-A10 and L cells were arown in stati;nary culture in McCoy’s 5a (modifiedy supplemented with Henes buffer (25 mM) and 10% fetal calf serum. Sixteen- hours prior to’ carrying out cell spreading experiments, stationary cultured cells were
were pretreated with spreading factor (spec. act. 65) at con-
MATERIALS
Exp Cell Res 102 (1976)
AND
METHODS
METHODS
Serum spreading
Table 1. Spreading of BHK21-13s cells with spreading factora 1st treatment* of the substratum
2nd treatmerit* of the substratum
Addition toC the medium
Expt 1 SF SF SF SF
Anti-SF
SF SF Expt 2 BSA BYG Anti-SF
SF Anti-SF Non-immune serum BSA BYG SF
Expt 3 SF Anti-BHK
Spreading
Complete Complete None None Complete Complete Complete
:FF
None None None
Anti-BHK ConA
None Complete None
a Details are in the text and “Methods”. b Additions were made as follows: SF (spec. act. 69, 31 pg/ml; BSA, 1 mglml; ByG, I mg/ml: Anti-SF, 5.24 mgjml; Anti-BHK, 1.68 mgjml. c Additions were made as follows: SF (spec. act. 65), 31 &ml; Anti-SF, 2.62 mg/ml; non-immune serum, 1: 10; BSA, I n&ml; B-JG, 1 mglml; Anti-BHK, 1.68 mg/ml; ConA, I mglml.
separated from their substrata with 0.25% trypsin (GIBCo) and placed into suspension culture in fresh erowth medium. Cellular adhesiveness and cell spreadin were measured bv ureviouslv used techniaues 12. 5f . Cells from the suspension cultures were collect& by centrifugation at 500 e for 2 min and washed and resusnended in attachment-medium (0.8 mM MgSOl .7H,O; 1 I6 mM NaCI: 5.4 mM KCI: 10.6 mM Na,HPO,: 5.6 mM Dglucose; 20 mM Hepes; final pH=7,0).‘Immediately prior to carrying out experiments, CaCl* was added to the incubations at a concentration of 0.5 mM. In cell attachment experiments, incubations of l.OI .5 x 106 cells in 3.0 ml of medium were carried out in Falcon no. 3013 culture flasks for the time periods indicated at 37°C. At the end of the incubations the flasks were subjected to shaking at I50 rpm on a New Brunswick R-2 reciprocating shaker (I in. stroke) for IO set at room temperature and the cells which were resuspended by this procedure (considered to be nonattached) were removed with a pipet. The turbidities of the starting cell suspensions and non-attached cell suspensions were determined at 640 nm with a Bausch and Lomb Spectronic 70 equipped with digital readout.
factor
53
Cell concentrations were calculated from a previously determined linear relationship between cell number and adsorbency. The per cent of cells attached in an experiment was calculated as the starting number of cells in an incubation minus the number of nonattached cells, divided by the starting number of cells. Control experiments in which the attached cells were recovered indicated the reliability of this technique. In cell spreading experiments, incubations of 0.51.0~ lo8 cells in 1.0 ml of medium were carried out in Falcon no. 3001 tissue culture dishes for 45 min at 37°C. At the end of the incubations the medium in the dishes was swirled and decanted and the extent of cell spreading was determined visually with a Zeiss Photoscope I phase contrast microscope equipped with a long working distance 16x objective. Cells were treated with N-ethylmaleimide (NEM), trypsin (Sigma) or L-I-tosylamide-2-phenylethyl chloromethyl ketone (TPCK), as follows. Harvested cells were placed in attachment medium at a concentration of lW/ml. NEM, trypsin, or TPCK (0.1 M dissolved in MeOH) were added at the concentrations indicated and the reactions were incubated at room temperature for 5 min (NEM or TPCK) or IO min (trvosin). The reaction with trypsin was stopped by the-addition of fetal calf serum (10%) to the incubations. The cells were centrifuged, washed, and resuspended in fresh attachment medium. Protein-coated substrata were prepared by incubating Falcon flasks or dishes for 5 min at room temperature with 1.0 ml of attachment medium containing sera or proteins as indicated. Subsequently, the nrotein-coated substrata were washed with deionized Hz0 [4]. Purification and chemical properties of spreading factor isolated from fetal calf serum will be described in detail elsewhere [l5]. Briefly, 200 ml of fetal calf serum were treated with phenylmethyl sulfonyl fluoride (0.2 mM) for 30 min at room temperature. The treated serum was then dialyzed exhaustively against 0.01 M potassium phosphate buffer (pH=S.O) and loaded onto a 2.5X23 cm DE-52 column previously equilibrated with the same buffer. The column was eluted with 500 ml of 0.1 M potassium phosphate (nH=8.0) followed by 500 ml of 0.35 M potassium phosphate (pH=S.O). The most active fractions, found in the 0.35 M eluate, were pooled and concentrated to 5.0 ml or less using an Amicon ultrafiltration cell with PM 30 membrane. The concentrated sample was loaded onto a 2.5~55 cm Bio-Gel A I.5 m column nreviouslv eauilibrated with 0.1 M potassium phosphate (pH=S:O) and the column was- eluted with the same buffer. The bulk of the activity eluted in a single peak with an approximate molecular weight of 5000@. Active fractions were pooled and concentrated as described above and stored at 0 to -20°C. No loss of activity was observed during storage up to several months. Protein concentrations were determined by the Lowry method [ 161. Spreading activity was measured by addition of serum or isolated proteins to be tested to soreading assays (as described above). The material to de tested was added in serial dilutions and one unit of activity was defined as the minimum amount of protein required to give complete cell spreading in the 45 min assay. The kind of results obtained by this Exp Cell
Res 102 (1976)
54
F. Grinnell
Table 2. Effect of inhibitors coated substrataa
on cell attachment
to fetal calf serum or spreading factor
% inhibition of attachmentb onto substrata coated with Inhibitor (cont.)
Period of attachment (min)
Fetal calf serum
Spreading factor
5 :i
63 78 83
67 78 91
5 IO 20 5 IO 20
18 56 44 47 66 58
41 60 23 65 56 30
N-Ethylmaleimide (0. I mM) L-l-Tosylamide2-phenyl ethyl chloromethyl ketone (0.02 mM) Trypsin (Sigma,
5 mslml)
LI The substrata were pretreated with spreading factor (spec. act. 65), 31 mg/ml, or fetal calf serum, 294 mg/ml. Cells were treated with the inhibitors as indicated. Incubations were carried out for the time periods shown. Other details are in the “Methods”. ’ % attachment (control)-% attachment (with inhibitor) x loo % attachment (control)
procedure are shown in fig. I. The assay gave essentially the same results whether the spreading factor to be tested was added to the medium or used to pretreat the substratum; however, if cells were first attached to the substratum in serum-free medium, higher concentrations of spreading factor were then required to induce complete spreading. Although this semiquantitative assay generally permitted reliable and reproducible determination of the activity, in some cases we wished to compare situations in which complete cell spreading did not occur. Under these circumstances, spreading activity was determined by visually observing 100-200 attached cells and estimating the percentage of spread cells: 100% (+++ +), 75 % (+++), 50% (++), 25% (+). This technique did not take into account the degree of cell spreading and can only be considered reliable for qualitative purposes. Using the assays described above, the specific activities of spreading factor from various stages of the purification were determined to be: uufractionated serum, 2-3; DE-52 concentrate, 18-30; A 1.5 m concentrate, 54-72. If unfractionated fetal calf serum was aged one month at 4”C, the specific activities of the serum and fractions purified from it were much less than those obtained using fresh serum. Antiserum against spreading factor was produced in rabbits by three injections at two week intervals of 1 mg of spreading factor (spec. act.=72) mixed 1: 1 with Freund’s complete adjuvant. The injections were at multiple interdermal sites. Blood was drawn 1 week following the last injection and allowed to clot overnight at 4°C. The IgG fraction was then isolated from the serum [ 171. Antiserum against BHK cells was produced by 1 Exp CellRes
102 (1976)
mg injections of isolated BHK21-13s cell membranes in a regimen the same as that described above. The membranes were isolated essentially by the method of Atkinson & Summers [18]. Cells were swollen at 4°C for 5 min in IO mM Tris-HCI (pH=8) and ruptured by three strokes with a Dounce homogenizer (Kontes Glass Co.). MgCls was added at a concentration of 50 mM and the homogenate layered on a discontinuous sucrose gradient (prepared in the same buffer): 10 ml of 30 % (w/w) sucrose and 10 ml of 45 % (w/w) sucrose. The gradients were centrifuged at 7 500 rpm for 30 min in a Spinco SW27 rotor. Plasma membranes collected at the 30%/45% interface. They were recovered and diluted 1: 3 with buffer and centrifuged for 20 min at 10000 rpm in a Sorvall SS34 rotor. The gradient was repeated a total of three times and the plasma membrane sheets and vesicles finally obtained were free of contamination as determined by electron microscopy. Antiserum produced against the BHK21-13s cell membranes was cytotoxic for whole cells at dilutions of 1: 16 or less. In a similar range it was also cytotoxic for BHK21C13 and BHK-py cells. The IgG fraction was isolated from the serum [ 171.
RESULTS Site of spreading factor activity The evidence presented in table 1 is a summary of experiments designed to test the site of action of purified fetal calf serum
Serum spreading factor
Table 3. Divalent spreading a Cation added to the medium (cont., mM)
MC& :: l:o ::oO CaCl, 0.2 0.5 1.0 MgCI,+CaCl, 0.2 (each) 0.5 (each)
cation dependence
of cell
Spreading onto substrata coated with Fetal calf serum
Spreading factor
n.d. + ++ +++ ++
++ +++ ++++ ++++ ++++
+ + +
+++ ++++ ++
+++ ++++
++++ ++++
a The substrata were pretreated with medium containing 294 &ml fetal calf serum or 31 /L&III spreading factor (spec. act. 65). Other details are in the “Methods” section. n.d., not done.
55
Cell spreading promoted by SF adsorbed to the substratum was completely inhibited by treatment with antibody (IgG fraction) that had been prepared against purified SF (Anti-SF). This was determined both by addition of Anti-SF (2.62 mglml) to the incubation medium or by a second treatment of the substratum with Anti-SF (5.24 mglml) following initial treatment of the substratum with SF (table 1, expt 1). In addition, blocking adsorbed SF with Anti-SF caused partial inhibition of cell attachment; however, the extent of this inhibition was quite variable from experiment to experiment. In some experiments substrata were pretreated with Anti-SF and then treated with SF; attachment occurred, but there was no cell spreading. Ligands directed against the cell surface -ConA or antibody (IgG fraction) prepared against isolated BHK21-13s cell membranes (Anti-BHK)-were also found to inhibit cell spreading when added to the incubation medium; however, they did not prevent cell attachment. This inhibition was directed at the cells and not at the substratum, since treatment of SF-coated substrata with AntiBHK (5.24 mglml) did not affect subsequent cell spreading (table 1, expt 3).
spreading factor (SF). Complete spreading of BHK21-13s cells was obtained either by adding SF to the attachment medium or by pre-treating the substratum with SF and making no further additions to the medium. Moreover, addition of other proteins to the medium such as bovine serum albumin Relationship of spreading factor to (BSA, 1 mg/ml) or whole bovine gamma globulins (BrG, 1 mg/ml) did not alter the spreading and attachment activity extent or pattern of cell spreading (table 1, associated with unfractionated expt 1). Complete cell spreading also oc- fetal calf serum curred to substrata treated with SF (31 pg/ Experiments similar to those described in ml) diluted by a high concentration of BSA table 1 were carried out with unfractionated (500 pg/ml). On the other hand, direct ad- fetal calf serum (1.86 mglml) instead of SF. sorption of BSA or ByG on to the sub- Complete cell spreading occurred with sestratum prevented subsequent cell spreadrum in the medium or adsorbed to the subing even after the addition of SF to the stratum. After the substratum was coated medium (table 1, expt 2). It is important to with serum, addition of BSA or ByG to the note that cell attachment to substrata medium did not affect subsequent spreadcoated with BSA or ByG occurred even ing; however, spreading was completely inhibited by the addition of Anti-SF. Also, though spreading did not. Exp Cell
Res 102 (1976)
56
F. Grinnell
Table 4. Identi$cation
of spreading factor
in various
Unfractionated
seraa
DE-52, 0.35 M eluate
Source of serum
Max. spreading obtained
Cont. required (r4dmU
Max. spreading obtained
Cont. required (dml)
Human Porcine Fetal calf Calf Rabbit Chicken
++ ++ ++++ +++ +++ ++
313 178 294 710 172 625
+++ ++++ ++++ ++++ ++++ +++
15 16 36 67 70 127
u The various sera were fractionated as described for fetal calf serum and added to the incubation Other details are in the text and “Methods”.
pretreatment of the substratum with BSA or ByG prevented spreading despite subsequent addition of serum to the medium. Finally, spreading onto serum-coated substrata was inhibited by the addition of cell surface ligands to the medium. It has been shown that cell attachment occurs by different mechanisms in the presence and absence of serum in the medium and that the effect of serum is a result of adsorption of serum components onto the substratum [2, 5, 7, lo]. Since cell attachment and cell spreading were experimentally separable, the question arose whether these functions were mediated by similar serum components. Comparative experiments on the attachment of cells to SF and serum-coated substrata were carried out as follows. We determined the time course of cell attachment to substrata coated with adsorbed spreading factor and found that there was a lag phase in attachment similar to that observed using substrata coated by proteins from unfractionated serum. In both experiments we used the minimal amounts of spreading factor (15.5 pg/ml) or unfractionated serum (372 pg/ml) that were necessary to promote complete BHK21-13s spreading. The lag phase in the time course of attachment is in Exp CellRes
IO2 (1976)
medium.
marked contrast to the rapid initial attachment that we have always observed in serum-free medium with untreated substrata. The effect of various inhibitors on cell attachment and spreading onto SF-coated substrata was also determined. We previously reported that treatment of cells with N-ethyl maleimide, trypsin, or active-sitedirected protease inhibitors resulted in an inhibition of cell attachment in serum-containing medium but not in serum-free medium [2, 41. The results in table 2 show that these reagents caused similar inhibition of cell attachment to substrata pretreated with Table 5. Spreading of various cell linesa pg/ml protein required in medium for complete cell spreading Cell tine
Fetal calf serum
Spreading factor
BHK-py BHK21-Cl3 BHK21-13s Lb
94 186 372 186
IS? is:5 15.5
HeLa CHO-Al0
744 372
::
D The specific activity of the spreading factor was 65. Other details are in the text and “Methods”. * Only partial cell spreading was obtained with L cells.
Serum spreading factor
57
complete cell spreading was obtained either by the addition of Mg*+ (1.0 mM) or Ca*+ (0.5 mM) to the incubation medium. On the other hand, complete cell spreading onto substrata coated with unfractionated serum required both Ca*+ and Mg*+ ions in the medium.
Analysis of spreading factor activity in different sera
2. Spreading of BHK21-13s cells. The substrata were pretreated with (a) attachment medium; (6) fetal calf serum, 350 pg/ml; or (c) spreading factor (spec. act. 69, 15.5 pg/ml. x230.
Fig.
SF or with unfractionated serum. Experiments carried out to 45 min demonstrated that in the presence of the inhibitors there was essentially no spreading of those cells which did become attached to the substrata. Divalent cation dependence of cell spreading
The experiments summarized in table 3 show that there was a marked difference in the divalent cation dependence of cell spreading onto substrata coated with SF compared with substrata coated by unfractionated serum. With SF on the substratum,
It was of interest to survey a variety of sera to determine whether the spreading factor was a component particular to fetal calf serum or a component common to many different sera. Unfractionated sera varied considerably in their abilities to promote spreading of BHK21-13s cells as shown in table 4. Also, not all sera promoted complete cell spreading, regardless of the concentration tested. In the case of porcine serum, addition at concentrations higher than 178 mg/ml resulted in a decreasing extent of cell spreading. The unfractionated sera were each subjected to chromatography on DEAE cellulose as described in “Methods”. The activity was found predominantly in the 0.35 M eluate as occurred with fetal calf serum. Experiments were carried out to determine the effect of Anti-SF on spreading promoted by the different sera. In these experiments, substrata were pretreated with the 0.35 M eluates of the different sera (concentrations shown in table 4, 4th column) and then cell spreading was determined in the presence of Anti-SF (2.62 mglml). AntiSF completely inhibited spreading of cells onto substrata pretreated with calf serum and porcine serum, as well as fetal calf serum. There was partial inhibition of spreading onto substrata coated with human serum and no inhibition with substrata coated by rabbit or chicken sera. Exp Cell
Res 102 (1976)
58
F. Grinnell
spreading was similar in most cases; however, L cells were not observed to undergo complete cell spreading either in the assay system or during normal cell growth. Using substrata coated with SF (at the minimal concentrations required to induce maximal cell spreading) it was found that addition of Anti-SF (2.62 mg/ml) to the medium inhibited the spreading of all the different cell lines. Addition of Anti-BHK (1.68 mglml) to the medium was found to inhibit the spreading of BHK-py and BHK21-Cl3 cells as well as BHK21-13s cells, but had no effect on the attachment or spreading of L, HeLa, and CHO-Al0 cells. Of particular significance was the observation that each cell line had its own peculiar shape into which it spread and that this shape was the same for spreading onto substrata coated with unfractionated serum or coated with SF. This is shown for BHK21-13s cells (fig. 2), and CHO-A10 cells (fig. 3). Model system studies Fig. 3. Spreading of CHO-Al0 cells. The substrata were pretreated with (a) attachment medium; (b) fetal calf serum, 700 pg/ml; or (c) spreading factor (spec. act. 65),62 pg/ml. x230.
A very simple model explaining cell spreading would be binding between a cell surface 0
Spreading of various cell lines with fetal calf serum and spreading factor
Since the spreading factor was originally detected and isolated using BHK21-13s cells, we decided to determine if it was specific for only that cell line, or if it worked with other cell lines as well. The data in table 5 summarize experiments with six different cell lines that we have tested, ineluding BHK21-13s. In every case, spreading required the addition of serum to the medium and also occurred with SF. The ratio of the amount of unfractionated serum to SF that was required for complete cell Exp CellRes
102 (1976)
0
0
BHK CELL
0
4. Model of cell spreading onto ligand coated substrata. Bonds can form between the acceptor on the cell surface and the ligand (L). (Left) the ligand is adsorbed to the substratum. Progressive attachments lead to cell spreading; (right) the ligand is in the medium as well as adsorbed to the substratum. Interactions between soluble ligand and the cell surface acceptors compete with and prevent interactions between the adsorbed ligand and the cell surface acceptor, thus inhibiting spreading. Fig.
Serum spreading factor
59
grammatically in fig. 4. Anti-BHK (1.68 mg/ ml) was adsorbed onto the substratum and cell attachment was then carried out in unsupplemented medium. BHK cells attached and spread as shown in fig. 5 ((a), compare with fig. 4, left). It is noteworthy that this cell spreading was not inhibited by the addition of Anti-SF to the medium. When AntiBHK was added to the attachment medium as well as adsorbed onto the substratum, there was no spreading of cells that became attached and many cell aggregates were observed (fig. 5b; compare with fig. 4, right).
DISCUSSION Fig. 5. Spreading of BHK21-13s cells onto AntiBHK coated substrata. (a) The substrata were pretreated with Anti-BHK, 1.68 mglml; (b) Anti-BHK was added to the medium at a concentration of 1.68 mglml.
x230.
receptor and a target protein adsorbed onto the substratum. However, if the target protein is free in the medium as well as on the substratum one might expect spreading to be inhibited, not promoted as shown dia-
__--
__-__
_~
Fig. 6. Model of cell spreading in serum containing medium. Bonds leading to cell spreading form between the receptor on the cell surface and the moditied A proteins (circles) adsorbed onto the substratum. No such bonds form between cell surface receptors and A proteins in solution (A) or B proteins in solution or adsorbed to the substratum (0).
The evidence presented in this paper needs to be discussed from several points of view: (i) the dependence of cell spreading upon a specific serum spreading factor; (ii) the relationship between cell-substratum interactions leading to cell attachment and those leading to cell spreading; and (iii) the relationship between spreading factor and other serum factors that influence the behavior of cultured cells. There appears to be a class of proteins found in different sera which are required for cell spreading onto a substratum and are similar with respect to their chromatographic properties on DEAE-cellulose. Some are also antigenically cross-reactive. The fetal calf serum factor (SF) was purified and its specificity of action was tested. SF had to be directly adsorbed to promote spreading. Prior adsorption of non-SF proteins inhibited spreading and in the experiment in which SF was indirectly adsorbed to the substratum, by reaction with previously adsorbed Anti-SF, there was also no spreading. Spreading factor probably does not represent more than about 2.5% of the total Exp CellRes
102 (1976)
60
F. Grinned
serum proteins based upon our current purification data. Nevertheless it apparently has a high affinity for the substratum and competes favorably against other serum proteins for substratum adsorption sites. For instance, exposing the substratum to a minimal amount of SF in the presence of a large excess of BSA did not diminish cell spreading promoted by SF. It is proposed that SF does not form the target with which cell surface receptors interact until after its adsorption to the substratum. Otherwise, one might expect a situation as described in figs 4 and 5 in which the adsorbed and soluble factors compete non-productively, preventing cell spreading instead of promoting it. It is known that most proteins adsorb to substrata and upon adsorption undergo changes in their conformations depending upon the chemical and physical properties of the substrata [19, 201. Activation mechanisms requiring protein adsorption have been described in the field of blood chemistry. For instance, in the intrinsic pathway of blood coagulation, the adsorption of factor XII results in its activation and the initiation of thrombogenesis [19, 211. In this context, the adsorption-activation of spreading factor becomes another example of a more generally known phenomenon which is schematically shown in fig. 6. Spreading factor (A) undergoes a change upon adsorption (triangles to circles) permitting its interaction with appropriate cell surface receptors. Other proteins (B) also adsorb but are unable to interact. The presence of spreading factor in a variety of different sera and its ability to induce normal spreading in a number of different cell lines suggests the generality of this phenomenon; however, no attempt has yet been made to determine if spreading ExpCeNRes
102 (1976)
factor plays any role in tissue organization in situ. It is noteworthy that several cell lines have been reported to undergo spreading in the absence of serum in the medium [lo, 22, 231. In these particular cases the cells may be able to deposit endogenouslysynthesized glycoproteins and/or protein polysaccharides on the substratum which serve as spreading factors. The synthesis and deposition of cell microexudates has been described and it has been proposed that cells can become attached to these substances [24-281. It was mentioned earlier that cell attachment occurs differently in the presence and absence of serum in the medium and that the special properties of attachment in serum-containing medium depend upon adsorption of serum components onto the substratum [2, 5, 7, lo]. Most of our previous studies have measured cell attachment from a kinetic point of view, without regard to cell spreading per se. We now emphasize that the results of experiments focused particularly on cell spreading, presented in this paper and a previous one [5], indicate a clear dichotomy between cell attachment and spreading in serum-containing medium. Attachment without spreading occurred onto serum-coated substrata: (i) in the absence of both Mg2+ and Ca2+ ions in the medium; (ii) if Anti-SF was added to the medium; and (iii) if cell surface ligands were added to the medium. In addition, some time ago we reported that centrifugation of cells against the substratum increased the rate of cell attachment but not cell spreading [3, 291. Given that attachment and spreading are separable processes, can anything be said about the relationships of cell surface receptors and adsorbed serum proteins involved? There is no clear answer to this question. If similar receptors were in-
Serum spreading
volved, it might have been expected that reagents which blocked receptors for attachment would also prevent spreading. This was the case for NEM, trypsin, and TPCK, which inhibited attachment and spreading, but was not the case for ConA and Anti-BHK which only inhibited spreading. If the same adsorbed serum protein was involved, then the properties of attachment to substrata coated by adsorbed SF or unfractionated serum ought to have been similar. This was true with regard to the time course of cell attachment and inhibitor studies; however, Anti-SF inhibited spreading onto both SF and serum-coated substrata but did not consistently inhibit attachment. Clearly, further studies are necessary to resolve the enigmatic questions raised. Finally, it should be pointed out that this is the first instance in which a serum factor has been isolated which specifically promotes cell spreading. Serum factors, required for cell attachment, have been reported by several laboratories [30-331, although numerous other investigators, including ourselves, have found that cell attachment actually occurs faster in serumfree medium than in serum-containing medium (e.g., [7, lo]). This apparent discrepancy may reflect how the measurements were made and how the cells were treated prior to analysis. Serum factors may have been required to reverse harmful effects of trypsinization if this reagent was used to remove cells from the substratum [34, 351, or to stabilize the cells if measurements were made following long-term incubations [lo]. Several growth factors have also been isolated from serum [36-381. Although spreading factor has no growth stimulating properties, the possibility that it potentiates the action of other serum growth factors cannot be discounted.
factor
61
Expert technical assistance was provided by MS Linda Forsythe and Mr Donald Hays. This research was supported by grants from the NIH CA14609 and GM21698.
REFERENCES 1. Grinnell, F, Milam, M & Srere, P A, J cell biol.56 (1973) 659. 2. Grinnell, F, Arch biochem biophys 160 (1974) 304. 3. - Ibid 165 (1974) 524. 4. - Ibid 169 (1975) 474. 5. - Exp cell res 97 (1975) 265. 6. Wolpert, L, Macpherson, I & Todd, I, Nature 223 (1%9) 513. 7. Nordling, S, Acta path01 microbial Stand, suppl. 192 (1%7) 1. 8. Price, P G, J membrane biol2 (1970) 300. 9. Witkowski, A & Brighton, W D, Exp cell res 70 (1972) 41. 10. Taylor, A C, Exp cell res, suppl. 8 (1961) 154. 11. Hayry, P, Myllyla, G, Saxen, E & Penttinen, K, Ann med exp fenn 44 (1966) 166. 12. Flaxman, B, Lutzner, M & VanScott, E, J cell biol 36 (1968) 406. 13. Brunk, U, Ericsson, J L E, Ponten, J & Westermark, B, Exp cell res 67 (1971) 407. 14. Revel, J P & Wolken, K, Exp cell res 78 (1973) 1. 15. Grinnell, F. In preparation. 16. Lowry, 0 H, Rosebrough, N J, Farr, A L & Randall, R J, J biol them 193 (1951) 265. 17. Fahey, J L, Methods in immunology (ed C A Williams & M W Chase) n. 321. Academic Press, New York (1967). 18. Atkinson, P H & Summers, D F, J biol them 246 (1971) 5162. 19. Vroman. L, Bull NY acad med 48 (1972) 302. 20. Baier, R E, Loeb, G I & Wallace, G T; Fed proc 30 (1971) 1523. 21. Austen, K F, Transp proc 6 (1974) 39. 22. Witkowski, J A & Brighton, W D, Exn cell res 68 (1971) 372. 23. Rajaraman, R, Rounds, D E, Yen, S P S &Rembaum, A, Exp cell res 88 (1974) 327. 24. Rosenberg, M D, Biophys j 1 (l%O) 137. 25. Maslow, D E & Weiss, L, Exp cell res 71 (1972) 204. 26. Poste, G, Greenham, L W, Mallucci, L, Reeve, P & Alexander, D J, Exp cell res 78 (1973) 303. 27. Culp, L A, J cell biol63 (1974) 71. 28. Weiss, L, Poste, G, MacKeamin, A & Willett, K, J cell biol64 (1975) 135. 29. Milam, M, Grinnell, F & Srere, P A, Nature new biol244 (1973) 83. 30. Fisher, H W, Puck, T T & Sato, G, hoc natl acad sci US 44 (1958) 4. 31. Lieberman, I &‘Ove, P, J cell bio1233 (1958) 637. Holmes. R. J cell biol32 (1%7) 297. ::. Klebe, R J; Nature 250 (1974) 248. 34: Hebb, C R & Chu, M W, Exp cell res 20 (1969) 453. 35. Weiss, L & Kapes, D L, Exp cell res 41(1966) 601. 36. Clarke, G D & Stoker, M G P, Growth control in Exp Cdl
Res 102 (1976)
62
F. Grinned
cell cultures (ed G E W Wolstenholme &J Knight) p. 17. Ciba Foundation Symposium (1971). 37. Paul, D, Lipton, A & Klinger, I, Proc natl acad sci us 68 (1971) 645. 38. Temin, H M, Pierson, R W Jr & Dulak, N C, Growth, nutrition and metabolism of cells in
Exp CeNRes 102 (1976)
culture (ed G H Rothblat & V Cristofalo) p. 50. Academic Press, New York (1972). Received February 3, 1976 Accepted May 4, 1976
Experimental
CELL Occurrence
Cell Research 102 (1976) 51-62
SPREADING
FACTOR
and Specificity
of Action
F. GRINNELL Department of Cell Biology, University of Texas, Southwestern Medical School, Dallas, TX 75235, USA
SUMMARY A factor required for spreading of substratum-attached baby hamster kidney cells (BHK), Chinese hamster ovary (CHO) cells, HeLa cells, and L cells has been isolated and purified from fetal calf serum. A similar factor has also been found in calf, porcine, human, rabbit, and chicken sera. The spreading factor was active when adsorbed to the substratum and prior adsorption of other proteins prevented cell spreading, regardless of the addition of spreading factor or unfractionated serum to the incubation medium. Antibody against the fetal calf spreading factor inhibited the spreading activity associated with unfractionated fetal calf serum and also the spreading activity associated with calf serum and porcine serum. In model system studies it was found that antibody against BHK cell surfaces induced cell spreading when the antibody was adsorbed to the substratum; when it was present in the incubation medium as well as on the substratum, cell spreading was not observed. The data are discussed in terms of the hypothesis that there is a specific serum factor which adsorbs to the substratum surface and is thereby activated, and which then forms the target for certain cell surface receptors. Interaction between adsorbed-activated factor and cell surface receptors leads to cell spreading.
The attachment of baby hamster kidney (BHK) cells in serum-containing medium to non-living substrata can best be described as a series of steps: adsorption of serum components onto the substratum, contact between the cells and substratum, initial attachment, and progressive attachments leading to cell spreading and an increased strength of cell attachment. Although there is a close interdependence of these events, especially contact and initial attachment, they have all been experimentally distinguished by biochemical techniques [l-5]. Some time ago, Wolpert et al. [6] reported that the spreading of BHK cells required the presence of serum in the medium. This has also been reported for HeLa cells [7] and L
cells [8], and for the normal morphology of cell spreading of MCR-5 cells [9]. It is likely that in serum-containing medium the substratum becomes coated with serum components and that attachment occurs to the adsorbed serum layer and not directly to the substratum [2, 4, 5, 7, 9, 10-141. Thus, we have become interested in determining whether the ability of cells to spread onto serum-coated substrata is a general property of protein-coated substrate or a specific property dependent upon certain serum components. In preliminary experiments we reported evidence suggesting that there was a requirement for specific serum components in the spreading of BHK cells [5]. We have now identified a specific Exp
Cell
Res
102 (1976)
52
F. Grinnell
Figs l-3,5. For further details, see “Methods”. Fig. 1. Assay of spreading factor. The substrata
centrations of (a) 3.1 pg/ml; (b) 6.2 Kg/ml; and (c) 15.5 pg/ml. X290.
spreading factor in serum and in this paper report on its biological occurrence and specificity.
Materials
trypsin (type XI, 7 300 units/mg), L-1-tosylamide-2uhenvlethvl chloromethvl ketone and phenylmethyl sulfonyl fluoride were purchased from Sigma Chemical Co., St Louis, MO. Whole bovine gamma globulins (fraction II) was a product of Miles Laboratories, Kankakee, Ill. Whatman DE-52, DEAE cellulose, was purchased from Reeve Angel, Clifton, N.J. Bio-Gel A 1.5 m was the product of Bio-Rad Laboratories, Richmond, Calif. Other reagent grade chemicals were obtained from Fisher Scientific, Houston, Texas.
Baby hamster kidney (BHK) cells which were suspension-culture adapted (BHK21-13s) were the gift of Dr Adrian Chappel, Communicable Disease Center, Atlanta, Ga. Polyoma-transformed BHK (BHK-py) cells were the gift of Dr Walter Eckhart, The Salk Institute, San Diego, Calif. Normal BHK (BHK21-C13) cells, NCTC clone 929 (L) cells, and HeLa cells were obtained from the American Type Culture Collection, Rockville, Md. Chinese Hamster Ovary A10 (CHOAlO) cells were the gift of Dr William Garrod, Southwestern Medical School, Dallas, Texas. Eagle’s Minimal Essential Medium (spinner modified), McCoy’s 5a medium (modified), fetal calf, porcine, calf, chicken and rabbit sera, MEM amino acids, MEM vitamins, and trypsin 0.25% were obtained from GIBCo, Grand Island, N.Y. Human serum was purchased from Microbiological Associates, Bethesda, Md. Bovine serum albumin (type F), (ConA), N-ethyl-maleimide,
BHK21-13s and BHK-py cells were grown in suspension culture and BHK-21-Cl3 cells were mown in stationary culture. The culture medium was Eagle’s MEM (spinner modified) with double the concentration of amino acids (except lxglutamine) and vitamins and supplemented with Hepes buffer (20 mM), 0.1 g/l ferric nitrate, 2.0 g/l dextrose, 10% tryptose phosphate broth, and 10% fetal calf serum. The final sodium bicarbonate concentration in the medium was 0.5 p/l. HeLa. CHO-A10 and L cells were arown in stati;nary culture in McCoy’s 5a (modifiedy supplemented with Henes buffer (25 mM) and 10% fetal calf serum. Sixteen- hours prior to’ carrying out cell spreading experiments, stationary cultured cells were
were pretreated with spreading factor (spec. act. 65) at con-
MATERIALS
Exp Cell Res 102 (1976)
AND
METHODS
METHODS
Serum spreading
Table 1. Spreading of BHK21-13s cells with spreading factora 1st treatment* of the substratum
2nd treatmerit* of the substratum
Addition toC the medium
Expt 1 SF SF SF SF
Anti-SF
SF SF Expt 2 BSA BYG Anti-SF
SF Anti-SF Non-immune serum BSA BYG SF
Expt 3 SF Anti-BHK
Spreading
Complete Complete None None Complete Complete Complete
:FF
None None None
Anti-BHK ConA
None Complete None
a Details are in the text and “Methods”. b Additions were made as follows: SF (spec. act. 69, 31 pg/ml; BSA, 1 mglml; ByG, I mg/ml: Anti-SF, 5.24 mgjml; Anti-BHK, 1.68 mgjml. c Additions were made as follows: SF (spec. act. 65), 31 &ml; Anti-SF, 2.62 mg/ml; non-immune serum, 1: 10; BSA, I n&ml; B-JG, 1 mglml; Anti-BHK, 1.68 mg/ml; ConA, I mglml.
separated from their substrata with 0.25% trypsin (GIBCo) and placed into suspension culture in fresh erowth medium. Cellular adhesiveness and cell spreadin were measured bv ureviouslv used techniaues 12. 5f . Cells from the suspension cultures were collect& by centrifugation at 500 e for 2 min and washed and resusnended in attachment-medium (0.8 mM MgSOl .7H,O; 1 I6 mM NaCI: 5.4 mM KCI: 10.6 mM Na,HPO,: 5.6 mM Dglucose; 20 mM Hepes; final pH=7,0).‘Immediately prior to carrying out experiments, CaCl* was added to the incubations at a concentration of 0.5 mM. In cell attachment experiments, incubations of l.OI .5 x 106 cells in 3.0 ml of medium were carried out in Falcon no. 3013 culture flasks for the time periods indicated at 37°C. At the end of the incubations the flasks were subjected to shaking at I50 rpm on a New Brunswick R-2 reciprocating shaker (I in. stroke) for IO set at room temperature and the cells which were resuspended by this procedure (considered to be nonattached) were removed with a pipet. The turbidities of the starting cell suspensions and non-attached cell suspensions were determined at 640 nm with a Bausch and Lomb Spectronic 70 equipped with digital readout.
factor
53
Cell concentrations were calculated from a previously determined linear relationship between cell number and adsorbency. The per cent of cells attached in an experiment was calculated as the starting number of cells in an incubation minus the number of nonattached cells, divided by the starting number of cells. Control experiments in which the attached cells were recovered indicated the reliability of this technique. In cell spreading experiments, incubations of 0.51.0~ lo8 cells in 1.0 ml of medium were carried out in Falcon no. 3001 tissue culture dishes for 45 min at 37°C. At the end of the incubations the medium in the dishes was swirled and decanted and the extent of cell spreading was determined visually with a Zeiss Photoscope I phase contrast microscope equipped with a long working distance 16x objective. Cells were treated with N-ethylmaleimide (NEM), trypsin (Sigma) or L-I-tosylamide-2-phenylethyl chloromethyl ketone (TPCK), as follows. Harvested cells were placed in attachment medium at a concentration of lW/ml. NEM, trypsin, or TPCK (0.1 M dissolved in MeOH) were added at the concentrations indicated and the reactions were incubated at room temperature for 5 min (NEM or TPCK) or IO min (trvosin). The reaction with trypsin was stopped by the-addition of fetal calf serum (10%) to the incubations. The cells were centrifuged, washed, and resuspended in fresh attachment medium. Protein-coated substrata were prepared by incubating Falcon flasks or dishes for 5 min at room temperature with 1.0 ml of attachment medium containing sera or proteins as indicated. Subsequently, the nrotein-coated substrata were washed with deionized Hz0 [4]. Purification and chemical properties of spreading factor isolated from fetal calf serum will be described in detail elsewhere [l5]. Briefly, 200 ml of fetal calf serum were treated with phenylmethyl sulfonyl fluoride (0.2 mM) for 30 min at room temperature. The treated serum was then dialyzed exhaustively against 0.01 M potassium phosphate buffer (pH=S.O) and loaded onto a 2.5X23 cm DE-52 column previously equilibrated with the same buffer. The column was eluted with 500 ml of 0.1 M potassium phosphate (nH=8.0) followed by 500 ml of 0.35 M potassium phosphate (pH=S.O). The most active fractions, found in the 0.35 M eluate, were pooled and concentrated to 5.0 ml or less using an Amicon ultrafiltration cell with PM 30 membrane. The concentrated sample was loaded onto a 2.5~55 cm Bio-Gel A I.5 m column nreviouslv eauilibrated with 0.1 M potassium phosphate (pH=S:O) and the column was- eluted with the same buffer. The bulk of the activity eluted in a single peak with an approximate molecular weight of 5000@. Active fractions were pooled and concentrated as described above and stored at 0 to -20°C. No loss of activity was observed during storage up to several months. Protein concentrations were determined by the Lowry method [ 161. Spreading activity was measured by addition of serum or isolated proteins to be tested to soreading assays (as described above). The material to de tested was added in serial dilutions and one unit of activity was defined as the minimum amount of protein required to give complete cell spreading in the 45 min assay. The kind of results obtained by this Exp Cell
Res 102 (1976)
54
F. Grinnell
Table 2. Effect of inhibitors coated substrataa
on cell attachment
to fetal calf serum or spreading factor
% inhibition of attachmentb onto substrata coated with Inhibitor (cont.)
Period of attachment (min)
Fetal calf serum
Spreading factor
5 :i
63 78 83
67 78 91
5 IO 20 5 IO 20
18 56 44 47 66 58
41 60 23 65 56 30
N-Ethylmaleimide (0. I mM) L-l-Tosylamide2-phenyl ethyl chloromethyl ketone (0.02 mM) Trypsin (Sigma,
5 mslml)
LI The substrata were pretreated with spreading factor (spec. act. 65), 31 mg/ml, or fetal calf serum, 294 mg/ml. Cells were treated with the inhibitors as indicated. Incubations were carried out for the time periods shown. Other details are in the “Methods”. ’ % attachment (control)-% attachment (with inhibitor) x loo % attachment (control)
procedure are shown in fig. I. The assay gave essentially the same results whether the spreading factor to be tested was added to the medium or used to pretreat the substratum; however, if cells were first attached to the substratum in serum-free medium, higher concentrations of spreading factor were then required to induce complete spreading. Although this semiquantitative assay generally permitted reliable and reproducible determination of the activity, in some cases we wished to compare situations in which complete cell spreading did not occur. Under these circumstances, spreading activity was determined by visually observing 100-200 attached cells and estimating the percentage of spread cells: 100% (+++ +), 75 % (+++), 50% (++), 25% (+). This technique did not take into account the degree of cell spreading and can only be considered reliable for qualitative purposes. Using the assays described above, the specific activities of spreading factor from various stages of the purification were determined to be: uufractionated serum, 2-3; DE-52 concentrate, 18-30; A 1.5 m concentrate, 54-72. If unfractionated fetal calf serum was aged one month at 4”C, the specific activities of the serum and fractions purified from it were much less than those obtained using fresh serum. Antiserum against spreading factor was produced in rabbits by three injections at two week intervals of 1 mg of spreading factor (spec. act.=72) mixed 1: 1 with Freund’s complete adjuvant. The injections were at multiple interdermal sites. Blood was drawn 1 week following the last injection and allowed to clot overnight at 4°C. The IgG fraction was then isolated from the serum [ 171. Antiserum against BHK cells was produced by 1 Exp CellRes
102 (1976)
mg injections of isolated BHK21-13s cell membranes in a regimen the same as that described above. The membranes were isolated essentially by the method of Atkinson & Summers [18]. Cells were swollen at 4°C for 5 min in IO mM Tris-HCI (pH=8) and ruptured by three strokes with a Dounce homogenizer (Kontes Glass Co.). MgCls was added at a concentration of 50 mM and the homogenate layered on a discontinuous sucrose gradient (prepared in the same buffer): 10 ml of 30 % (w/w) sucrose and 10 ml of 45 % (w/w) sucrose. The gradients were centrifuged at 7 500 rpm for 30 min in a Spinco SW27 rotor. Plasma membranes collected at the 30%/45% interface. They were recovered and diluted 1: 3 with buffer and centrifuged for 20 min at 10000 rpm in a Sorvall SS34 rotor. The gradient was repeated a total of three times and the plasma membrane sheets and vesicles finally obtained were free of contamination as determined by electron microscopy. Antiserum produced against the BHK21-13s cell membranes was cytotoxic for whole cells at dilutions of 1: 16 or less. In a similar range it was also cytotoxic for BHK21C13 and BHK-py cells. The IgG fraction was isolated from the serum [ 171.
RESULTS Site of spreading factor activity The evidence presented in table 1 is a summary of experiments designed to test the site of action of purified fetal calf serum
Serum spreading factor
Table 3. Divalent spreading a Cation added to the medium (cont., mM)
MC& :: l:o ::oO CaCl, 0.2 0.5 1.0 MgCI,+CaCl, 0.2 (each) 0.5 (each)
cation dependence
of cell
Spreading onto substrata coated with Fetal calf serum
Spreading factor
n.d. + ++ +++ ++
++ +++ ++++ ++++ ++++
+ + +
+++ ++++ ++
+++ ++++
++++ ++++
a The substrata were pretreated with medium containing 294 &ml fetal calf serum or 31 /L&III spreading factor (spec. act. 65). Other details are in the “Methods” section. n.d., not done.
55
Cell spreading promoted by SF adsorbed to the substratum was completely inhibited by treatment with antibody (IgG fraction) that had been prepared against purified SF (Anti-SF). This was determined both by addition of Anti-SF (2.62 mglml) to the incubation medium or by a second treatment of the substratum with Anti-SF (5.24 mglml) following initial treatment of the substratum with SF (table 1, expt 1). In addition, blocking adsorbed SF with Anti-SF caused partial inhibition of cell attachment; however, the extent of this inhibition was quite variable from experiment to experiment. In some experiments substrata were pretreated with Anti-SF and then treated with SF; attachment occurred, but there was no cell spreading. Ligands directed against the cell surface -ConA or antibody (IgG fraction) prepared against isolated BHK21-13s cell membranes (Anti-BHK)-were also found to inhibit cell spreading when added to the incubation medium; however, they did not prevent cell attachment. This inhibition was directed at the cells and not at the substratum, since treatment of SF-coated substrata with AntiBHK (5.24 mglml) did not affect subsequent cell spreading (table 1, expt 3).
spreading factor (SF). Complete spreading of BHK21-13s cells was obtained either by adding SF to the attachment medium or by pre-treating the substratum with SF and making no further additions to the medium. Moreover, addition of other proteins to the medium such as bovine serum albumin Relationship of spreading factor to (BSA, 1 mg/ml) or whole bovine gamma globulins (BrG, 1 mg/ml) did not alter the spreading and attachment activity extent or pattern of cell spreading (table 1, associated with unfractionated expt 1). Complete cell spreading also oc- fetal calf serum curred to substrata treated with SF (31 pg/ Experiments similar to those described in ml) diluted by a high concentration of BSA table 1 were carried out with unfractionated (500 pg/ml). On the other hand, direct ad- fetal calf serum (1.86 mglml) instead of SF. sorption of BSA or ByG on to the sub- Complete cell spreading occurred with sestratum prevented subsequent cell spreadrum in the medium or adsorbed to the subing even after the addition of SF to the stratum. After the substratum was coated medium (table 1, expt 2). It is important to with serum, addition of BSA or ByG to the note that cell attachment to substrata medium did not affect subsequent spreadcoated with BSA or ByG occurred even ing; however, spreading was completely inhibited by the addition of Anti-SF. Also, though spreading did not. Exp Cell
Res 102 (1976)
56
F. Grinnell
Table 4. Identi$cation
of spreading factor
in various
Unfractionated
seraa
DE-52, 0.35 M eluate
Source of serum
Max. spreading obtained
Cont. required (r4dmU
Max. spreading obtained
Cont. required (dml)
Human Porcine Fetal calf Calf Rabbit Chicken
++ ++ ++++ +++ +++ ++
313 178 294 710 172 625
+++ ++++ ++++ ++++ ++++ +++
15 16 36 67 70 127
u The various sera were fractionated as described for fetal calf serum and added to the incubation Other details are in the text and “Methods”.
pretreatment of the substratum with BSA or ByG prevented spreading despite subsequent addition of serum to the medium. Finally, spreading onto serum-coated substrata was inhibited by the addition of cell surface ligands to the medium. It has been shown that cell attachment occurs by different mechanisms in the presence and absence of serum in the medium and that the effect of serum is a result of adsorption of serum components onto the substratum [2, 5, 7, lo]. Since cell attachment and cell spreading were experimentally separable, the question arose whether these functions were mediated by similar serum components. Comparative experiments on the attachment of cells to SF and serum-coated substrata were carried out as follows. We determined the time course of cell attachment to substrata coated with adsorbed spreading factor and found that there was a lag phase in attachment similar to that observed using substrata coated by proteins from unfractionated serum. In both experiments we used the minimal amounts of spreading factor (15.5 pg/ml) or unfractionated serum (372 pg/ml) that were necessary to promote complete BHK21-13s spreading. The lag phase in the time course of attachment is in Exp CellRes
IO2 (1976)
medium.
marked contrast to the rapid initial attachment that we have always observed in serum-free medium with untreated substrata. The effect of various inhibitors on cell attachment and spreading onto SF-coated substrata was also determined. We previously reported that treatment of cells with N-ethyl maleimide, trypsin, or active-sitedirected protease inhibitors resulted in an inhibition of cell attachment in serum-containing medium but not in serum-free medium [2, 41. The results in table 2 show that these reagents caused similar inhibition of cell attachment to substrata pretreated with Table 5. Spreading of various cell linesa pg/ml protein required in medium for complete cell spreading Cell tine
Fetal calf serum
Spreading factor
BHK-py BHK21-Cl3 BHK21-13s Lb
94 186 372 186
IS? is:5 15.5
HeLa CHO-Al0
744 372
::
D The specific activity of the spreading factor was 65. Other details are in the text and “Methods”. * Only partial cell spreading was obtained with L cells.
Serum spreading factor
57
complete cell spreading was obtained either by the addition of Mg*+ (1.0 mM) or Ca*+ (0.5 mM) to the incubation medium. On the other hand, complete cell spreading onto substrata coated with unfractionated serum required both Ca*+ and Mg*+ ions in the medium.
Analysis of spreading factor activity in different sera
2. Spreading of BHK21-13s cells. The substrata were pretreated with (a) attachment medium; (6) fetal calf serum, 350 pg/ml; or (c) spreading factor (spec. act. 69, 15.5 pg/ml. x230.
Fig.
SF or with unfractionated serum. Experiments carried out to 45 min demonstrated that in the presence of the inhibitors there was essentially no spreading of those cells which did become attached to the substrata. Divalent cation dependence of cell spreading
The experiments summarized in table 3 show that there was a marked difference in the divalent cation dependence of cell spreading onto substrata coated with SF compared with substrata coated by unfractionated serum. With SF on the substratum,
It was of interest to survey a variety of sera to determine whether the spreading factor was a component particular to fetal calf serum or a component common to many different sera. Unfractionated sera varied considerably in their abilities to promote spreading of BHK21-13s cells as shown in table 4. Also, not all sera promoted complete cell spreading, regardless of the concentration tested. In the case of porcine serum, addition at concentrations higher than 178 mg/ml resulted in a decreasing extent of cell spreading. The unfractionated sera were each subjected to chromatography on DEAE cellulose as described in “Methods”. The activity was found predominantly in the 0.35 M eluate as occurred with fetal calf serum. Experiments were carried out to determine the effect of Anti-SF on spreading promoted by the different sera. In these experiments, substrata were pretreated with the 0.35 M eluates of the different sera (concentrations shown in table 4, 4th column) and then cell spreading was determined in the presence of Anti-SF (2.62 mglml). AntiSF completely inhibited spreading of cells onto substrata pretreated with calf serum and porcine serum, as well as fetal calf serum. There was partial inhibition of spreading onto substrata coated with human serum and no inhibition with substrata coated by rabbit or chicken sera. Exp Cell
Res 102 (1976)
58
F. Grinnell
spreading was similar in most cases; however, L cells were not observed to undergo complete cell spreading either in the assay system or during normal cell growth. Using substrata coated with SF (at the minimal concentrations required to induce maximal cell spreading) it was found that addition of Anti-SF (2.62 mg/ml) to the medium inhibited the spreading of all the different cell lines. Addition of Anti-BHK (1.68 mglml) to the medium was found to inhibit the spreading of BHK-py and BHK21-Cl3 cells as well as BHK21-13s cells, but had no effect on the attachment or spreading of L, HeLa, and CHO-Al0 cells. Of particular significance was the observation that each cell line had its own peculiar shape into which it spread and that this shape was the same for spreading onto substrata coated with unfractionated serum or coated with SF. This is shown for BHK21-13s cells (fig. 2), and CHO-A10 cells (fig. 3). Model system studies Fig. 3. Spreading of CHO-Al0 cells. The substrata were pretreated with (a) attachment medium; (b) fetal calf serum, 700 pg/ml; or (c) spreading factor (spec. act. 65),62 pg/ml. x230.
A very simple model explaining cell spreading would be binding between a cell surface 0
Spreading of various cell lines with fetal calf serum and spreading factor
Since the spreading factor was originally detected and isolated using BHK21-13s cells, we decided to determine if it was specific for only that cell line, or if it worked with other cell lines as well. The data in table 5 summarize experiments with six different cell lines that we have tested, ineluding BHK21-13s. In every case, spreading required the addition of serum to the medium and also occurred with SF. The ratio of the amount of unfractionated serum to SF that was required for complete cell Exp CellRes
102 (1976)
0
0
BHK CELL
0
4. Model of cell spreading onto ligand coated substrata. Bonds can form between the acceptor on the cell surface and the ligand (L). (Left) the ligand is adsorbed to the substratum. Progressive attachments lead to cell spreading; (right) the ligand is in the medium as well as adsorbed to the substratum. Interactions between soluble ligand and the cell surface acceptors compete with and prevent interactions between the adsorbed ligand and the cell surface acceptor, thus inhibiting spreading. Fig.
Serum spreading factor
59
grammatically in fig. 4. Anti-BHK (1.68 mg/ ml) was adsorbed onto the substratum and cell attachment was then carried out in unsupplemented medium. BHK cells attached and spread as shown in fig. 5 ((a), compare with fig. 4, left). It is noteworthy that this cell spreading was not inhibited by the addition of Anti-SF to the medium. When AntiBHK was added to the attachment medium as well as adsorbed onto the substratum, there was no spreading of cells that became attached and many cell aggregates were observed (fig. 5b; compare with fig. 4, right).
DISCUSSION Fig. 5. Spreading of BHK21-13s cells onto AntiBHK coated substrata. (a) The substrata were pretreated with Anti-BHK, 1.68 mglml; (b) Anti-BHK was added to the medium at a concentration of 1.68 mglml.
x230.
receptor and a target protein adsorbed onto the substratum. However, if the target protein is free in the medium as well as on the substratum one might expect spreading to be inhibited, not promoted as shown dia-
__--
__-__
_~
Fig. 6. Model of cell spreading in serum containing medium. Bonds leading to cell spreading form between the receptor on the cell surface and the moditied A proteins (circles) adsorbed onto the substratum. No such bonds form between cell surface receptors and A proteins in solution (A) or B proteins in solution or adsorbed to the substratum (0).
The evidence presented in this paper needs to be discussed from several points of view: (i) the dependence of cell spreading upon a specific serum spreading factor; (ii) the relationship between cell-substratum interactions leading to cell attachment and those leading to cell spreading; and (iii) the relationship between spreading factor and other serum factors that influence the behavior of cultured cells. There appears to be a class of proteins found in different sera which are required for cell spreading onto a substratum and are similar with respect to their chromatographic properties on DEAE-cellulose. Some are also antigenically cross-reactive. The fetal calf serum factor (SF) was purified and its specificity of action was tested. SF had to be directly adsorbed to promote spreading. Prior adsorption of non-SF proteins inhibited spreading and in the experiment in which SF was indirectly adsorbed to the substratum, by reaction with previously adsorbed Anti-SF, there was also no spreading. Spreading factor probably does not represent more than about 2.5% of the total Exp CellRes
102 (1976)
60
F. Grinned
serum proteins based upon our current purification data. Nevertheless it apparently has a high affinity for the substratum and competes favorably against other serum proteins for substratum adsorption sites. For instance, exposing the substratum to a minimal amount of SF in the presence of a large excess of BSA did not diminish cell spreading promoted by SF. It is proposed that SF does not form the target with which cell surface receptors interact until after its adsorption to the substratum. Otherwise, one might expect a situation as described in figs 4 and 5 in which the adsorbed and soluble factors compete non-productively, preventing cell spreading instead of promoting it. It is known that most proteins adsorb to substrata and upon adsorption undergo changes in their conformations depending upon the chemical and physical properties of the substrata [19, 201. Activation mechanisms requiring protein adsorption have been described in the field of blood chemistry. For instance, in the intrinsic pathway of blood coagulation, the adsorption of factor XII results in its activation and the initiation of thrombogenesis [19, 211. In this context, the adsorption-activation of spreading factor becomes another example of a more generally known phenomenon which is schematically shown in fig. 6. Spreading factor (A) undergoes a change upon adsorption (triangles to circles) permitting its interaction with appropriate cell surface receptors. Other proteins (B) also adsorb but are unable to interact. The presence of spreading factor in a variety of different sera and its ability to induce normal spreading in a number of different cell lines suggests the generality of this phenomenon; however, no attempt has yet been made to determine if spreading ExpCeNRes
102 (1976)
factor plays any role in tissue organization in situ. It is noteworthy that several cell lines have been reported to undergo spreading in the absence of serum in the medium [lo, 22, 231. In these particular cases the cells may be able to deposit endogenouslysynthesized glycoproteins and/or protein polysaccharides on the substratum which serve as spreading factors. The synthesis and deposition of cell microexudates has been described and it has been proposed that cells can become attached to these substances [24-281. It was mentioned earlier that cell attachment occurs differently in the presence and absence of serum in the medium and that the special properties of attachment in serum-containing medium depend upon adsorption of serum components onto the substratum [2, 5, 7, lo]. Most of our previous studies have measured cell attachment from a kinetic point of view, without regard to cell spreading per se. We now emphasize that the results of experiments focused particularly on cell spreading, presented in this paper and a previous one [5], indicate a clear dichotomy between cell attachment and spreading in serum-containing medium. Attachment without spreading occurred onto serum-coated substrata: (i) in the absence of both Mg2+ and Ca2+ ions in the medium; (ii) if Anti-SF was added to the medium; and (iii) if cell surface ligands were added to the medium. In addition, some time ago we reported that centrifugation of cells against the substratum increased the rate of cell attachment but not cell spreading [3, 291. Given that attachment and spreading are separable processes, can anything be said about the relationships of cell surface receptors and adsorbed serum proteins involved? There is no clear answer to this question. If similar receptors were in-
Serum spreading
volved, it might have been expected that reagents which blocked receptors for attachment would also prevent spreading. This was the case for NEM, trypsin, and TPCK, which inhibited attachment and spreading, but was not the case for ConA and Anti-BHK which only inhibited spreading. If the same adsorbed serum protein was involved, then the properties of attachment to substrata coated by adsorbed SF or unfractionated serum ought to have been similar. This was true with regard to the time course of cell attachment and inhibitor studies; however, Anti-SF inhibited spreading onto both SF and serum-coated substrata but did not consistently inhibit attachment. Clearly, further studies are necessary to resolve the enigmatic questions raised. Finally, it should be pointed out that this is the first instance in which a serum factor has been isolated which specifically promotes cell spreading. Serum factors, required for cell attachment, have been reported by several laboratories [30-331, although numerous other investigators, including ourselves, have found that cell attachment actually occurs faster in serumfree medium than in serum-containing medium (e.g., [7, lo]). This apparent discrepancy may reflect how the measurements were made and how the cells were treated prior to analysis. Serum factors may have been required to reverse harmful effects of trypsinization if this reagent was used to remove cells from the substratum [34, 351, or to stabilize the cells if measurements were made following long-term incubations [lo]. Several growth factors have also been isolated from serum [36-381. Although spreading factor has no growth stimulating properties, the possibility that it potentiates the action of other serum growth factors cannot be discounted.
factor
61
Expert technical assistance was provided by MS Linda Forsythe and Mr Donald Hays. This research was supported by grants from the NIH CA14609 and GM21698.
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cell cultures (ed G E W Wolstenholme &J Knight) p. 17. Ciba Foundation Symposium (1971). 37. Paul, D, Lipton, A & Klinger, I, Proc natl acad sci us 68 (1971) 645. 38. Temin, H M, Pierson, R W Jr & Dulak, N C, Growth, nutrition and metabolism of cells in
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culture (ed G H Rothblat & V Cristofalo) p. 50. Academic Press, New York (1972). Received February 3, 1976 Accepted May 4, 1976