Download the effects of egta and trypsin on the serum requirements for cell

J. Cell Sci. 40, 271-279 (1979)
Printed in Great Britain © Company of Biologists Limited 1979
271
THE EFFECTS OF EGTA AND TRYPSIN ON
THE SERUM REQUIREMENTS FOR CELL
ATTACHMENT TO COLLAGEN
SETH L. SCHOR
Cancer Research Campaign (Department of Medical Oncology),
University of Manchester and Christie Hospital and Holt Radium
Wilmslow Road, Manchester 20 <)BX, England
Institute,
SUMMARY
Cells growing on plastic or glass surfaces in vitro may be brought into suspension by proteases
(e.g. trypsin) or chelating agents (e.g. EGTA). Trypsin and EGTA remove different quantities
and types of molecules from cell surfaces. Previous studies have revealed that when confluent
cultures of either BHK or PyBHK cells are brought into suspension by exposure to trypsin,
foetal calf serum (or fibronectin) is required for cell attachment to films of denatured type I
collagen, but not to 3-dimensional gels of native collagen fibres. In this communication the
serum requirements for the attachment of BHK and PyBHK cells to collagen substrata have
been examined as a function of (a) the method used to prepare the cell suspension (EGTA or
trypsin), and (b) cell density. Data are presented consistent with the view that cell surfaceassociated fibronectin is able to mediate cell attachment directly to films of denatured collagen.
INTRODUCTION
Grobstein (1953) first proposed that the extracellular matrix plays an important
role in the control of cell behaviour during ontogeny. Collagen is a major constituent
of the extracellular matrix and there is a growing body of information, in support of
Grobstein's hypothesis, indicating that collagen can have a marked effect in vitro on
cell differentiation (Konigsberg & Hauschka, 1965; Meier & Hay, 1974; Kosher &
Church, 1975), proliferation (Svotelis, Foard & Bang, 1974; Liotta et al. 1978), and
migration (Bard & Hay, 1975; Stenn, Mackie & Roll, 1979). Although the means by
which collagen exerts these effects on cell behaviour are not understood, it is clear
that cell attachment to the collagen substratum is required (Meier & Hay, 1975; Lash
& Vasan, 1977; Bunge & Bunge, 1978), and it is for this reason that studies dealing
with the mechanism of cell attachment to collagen assume a particular importance.
Klebe (1974) observed that serum was required for the attachment of SV3T3
cells to films of type I collagen (pretreated with 8 M urea) and was able to isolate
the glycoprotein from serum responsible for this activity. A similar glycoprotein
capable of mediating cell attachment to films of collagen was isolated from the surface
of BHK cells (Pearlstein, 1976) and subsequent work in a number of laboratories
has demonstrated that the serum-derived and cell surface-derived glycoproteins
described by these authors are immunologically indistinguishable and now collectively
referred to as fibronectin (Yamada & Olden, 1978; Vaheri & Mosher, 1978; Grinnell
18-2
272
S. L. Schor
& Hays, 1978). More recent studies have indicated that serum or fibronectin is
actually only required for the attachment of cells to films of denatured type I collagen
and not to native collagen fibres (Schor & Court, 1979; Grinnell & Minter, 1978).
All of the above studies concerned with the requirements for cell attachment to
collagen have employed trypsin in preparing the cell suspension to be used in the
attachment assay. Cell suspensions can also be prepared using chelating agents such
as EDTA (ethylenediamine tetra-acetic acid) and EGTA (ethyleneglycol-bis-(/?aminoethyl ether) N,N'-tetra-acetic acid). Chelating agents remove different amounts
and types of surface-associated molecules from cells than trypsin (Snow & Allen,
1970; Codington, Sanford & Jeanloz, 1970; Huet & Herzberg, 1973; Kelley &
Lauer, 1975; Anghileri & Dermeitzel, 1976) and cell suspensions prepared by these
2 means display differences in the kinetics of cell attachment to cellular monolayers
(Brugmans, Cassiman & Van den Berghe, 1978), cell aggregation (Cassiman &
Bernfield, 1974), and cell agglutination by lectins (Burger, 1969). Trypsin may also
be internalized by the cell and its action therefore not restricted to the cell surface
(Hodges, Livingston & Franks, 1973).
The purpose of this study is to compare the effects of EGTA and trypsin on the
attachment of BHK and PyBHK cells tofilmsof denatured collagen and 3-dimensional
gels of native collagen fibres.
MATERIALS AND METHODS
Cells and culture conditions
BHK21/C13 and polyoma virus-transformed cells (PyBHK) were grown in Eagle's minimal
essential medium (MEM) supplemented with 10% foetal calf serum, 1 mM sodium pyruvate,
2 mM glutamine, non-essential amino acids (Gibco Bio Cult, Ltd., Glasgow, cat. no. 114) and
100 units/ml penicillin and streptomycin. Stock cultures growing in 90-mm plastic Petri
dishes (Gibco Bio Cult, Ltd., Uxbridge, cat no. 1415) were subcultured once a week and medium
changed 3 times a week. Confluent cultures contained 4-6 x 10* cells/dish and subconfluent
cultures 4-6 x io 5 cells/dish at harvesting.
Preparation of collagen substrata
An aqueous solution of type I tropocollagen was prepared from rat tail tendons as described
by Elsdale & Bard (1972). The optical density of the tropocollagen solution was measured at
230 nm and the concentration of tropocollagen calculated using a calibration curve prepared
from standard solutions of freeze-dried rat tail tendon collagen extracted in the same manner.
The concentration of tropocollagen in the aqueous stock solutions was adjusted to between
2'0 and 2'5 mg/ml prior to use.
Three-dimensional gels of native collagen fibres were prepared in 35-mm plastic tissue
culture Petri dishes (Gibco Bio Cult, Ltd., Uxbridge, cat. no. 53066), from the aqueous
tropocollagen stock solution according to Elsdale & Bard (1972). Heat-denatured collagen
was prepared by incubating the tropocollagen solution at 60 °C for 30 min and quickly lowering
the temperature by immersion into an ice bath. Films of heat-denatured collagen were made
by covering the bottoms of 35-mm plastic tissue culture Petri dishes with the solution of heatdenatured collagen, removing excess liquid with a Pasteur pipette and incubating the dishes
for 20 min in a dessicator containing an open beaker of concentrated ammonia. The films were
then air-dried and washed 5 times with distilled water before use. The arrangement of collagen
molecules within these films is discussed in Schor & Court (1979).
Cell attachment to collagen
273
Preparation of single cell suspensions and determinations of the kinetics of cell attachment
Separate stock solutions of 2 mM EGTA and 025 % crystalline trypsin were prepared in
Dulbecco's phosphate-buffered saline (PBS). Cell cultures were washed 3 times with Hanks'
balanced salt solution and then incubated at 37 CC with either (a) 4 ml of 2 mM EGTA for
20 min, or (b) 4 ml of 2 mM EGTA for 20 min, followed by the addition of 1 ml of 0-25 %
trypsin (giving a final concentration of 0-05 % trypsin) and continuing the incubation for an
additional 3 min. One millilitre of 0-25 % soybean trypsin inhibitor was then added to all
dishes, the cells pipetted several times to produce a uniform single cell suspension, centrifuged
at 300 g for s min, resuspended in serum-free MEM and the concentration adjusted to approximately 1 x 10s cells/ml. Untreated plastic tissue culture dishes and dishes containing the
collagen substrata were incubated for 1 h at 37 °C with 1 ml of either serum-free MEM or
MEM containing 10% foetal calf serum. At the beginning of the experiment 1 ml of cell suspension was added to these dishes, thus giving a cell suspension in either serum-free MEM or
MEM containing 5 % foetal calf serum. The dishes were kept at 37 °C in a humidified CO,
incubator and the number of unattached cells determined at the beginning of the experiment
(i.e., total number of cells/dish) and at various times thereafter by transferring the medium
(2 ml) from each dish to a vial containing 5 ml of' Isoton' (Coulter Electronics, Ltd., Harpenden, Herts.), gently washing the dishes 3 times with Hanks' balanced salt solution (3 ml in
total), adding these washes to the vial and finally determining the number of cells recovered
in this manner with a Coulter electronic particle counter. The number of attached cells at
any time could then be calculated by subtracting the number of unattached cells from the
total cell number per dish. Data are expressed as the percentage of cells attached. The kinetics
of cell attachment must be calculated in this manner since cells attached to native collagen
fibres are not completely removed by exposure to trypsin (Schor & Court, 1979).
Chemicals
Crystalline trypsin (T-8253), soybean trypsin inhibitor (T-9003) and EGTA (E-4378)
were all obtained from Sigma Chemical Co., London.
RESULTS
BHK and PyBHK cells brought into suspension by trypsin have been reported
to require the presence of serum (or fibronectin) for attachment to films of denatured
type I collagen, but not to native collagen fibres (Schor & Court, 1979; Grinnell &
Minter, 1978). In the experiments presented here, confluent cultures of BHK and
PyBHK cells were brought into suspension by exposure either to EGTA only or
EGTA and trypsin, and then used to measure the kinetics of cell attachment to
collagen substrata in the presence of serum-free growth medium or growth medium
containing 5 % foetal calf serum. The results obtained in a representative experiment
with confluent cultures of PyBHK cells are shown in Fig. 1. The kinetics of attachment to the different substrata of PyBHK cells treated with EGTA only are indistinguishable from those obtained with cells treated with trypsin. Different results are
obtained with confluent cultures of BHK cells (Fig. 2). In this case cells treated with
EGTA only no longer require serum for attachment to films of denatured collagen.
The results obtained in 5 such experiments are presented in Table 1. Data are
expressed as (a) the mean + S.D. of the percentage of cells attached to the various
substrata after 120 min of incubation, and (b) the difference between the percentage
attached to each substratum in the presence and absence of serum. These results
S. L. Schor
274
100 I-
A
.,—o
60
o
120
60
120
Fig. i. The attachment of PyBHK cells from confluent cultures to collagen substrata. PyBHK cells were harvested from confluent cultures by exposure either to
EGTA (A-C) only or EGTA + trypsin (D-F), as described in Materials and methods.
These cells were then used to measure the kinetics of cell attachment to plastic tissue
culture dishes (A, D), films of denatured type I collagen (B, H) and 3-dimensional gels
of native collagen fibres (c, F) either in serum-free MEM (O) or MEM containing
5 % foetal calf serum ( • ) . Data are expressed as the percentage of cells attached
after 30, 60 and 120 min of incubation.
100
60
120
Fig. 2. The attachment of BHK cells from confluent cultures to collagen substrata.
BHK cells were harvested from confluent cultures by exposure either to EGTA (A—c)
only or EGTA + trypsin (D—F), as described in Materials and methods. These cells
were then used to measure the kinetics of cell attachment to plastic tissue culture
dishes (A, D), films of denatured type I collagen (B, E) and 3-dimensional gels of
native collagen fibres (c, F) either in serum-free MEM (O) or MEM containing
5 % foetal calf serum ( • ) . Data are expressed as the percentage of cells attached after
30, 60 and 120 min of incubation.
Cell attachment to collagen
275
indicate that the attachment of confluent BHK cells treated with EGTA only to films
of denatured collagen is not serum-dependent, whereas the attachment of cells removed by EGTA and trypsin shows a significant serum-dependence, comparable to
that observed with PyBHK cells treated with either EGTA or EGTA and trypsin.
The attachment of BHK and PyBHK cells to gels of native collagen fibres is not
dependent on serum under any of the conditions tested.
Table 1. The attachment of PyBHK and BHK cells from confluent cultures
to collagen substrata
EGTA
Cell
PyBKH
Substratum
Plastic
Denatured
collagen films
Native collagen
gels
BHK
A attached
A attached
( + serum) —
( + serum) —
Serum % attachment ( — serum) % attachment ( — serum)
+
847 ±o-6
+
—
91-4121
833 ±4-8
36-0 ±2-9
83-9 ± 9 7
+
Plastic
Denatured
collagen films
Native collagen
gels
EGTA + trypsin
}
/
}
84-8 ±6-5
82-2 ± 7 0
87-916-1 }
8i-3±8-o
84-8 ±2-9
83-3i3-8
47'3 ±0-2
}
{777 ±6-9
}
;}
\ 91 "2,14*0
[ 75 " 6 l i i "
) 4 O - o l io-
13-5 ±5-8
35-2! 11-5
-09±32
{734±3°
-5717-5
-9-il4-8
{91-4157
(75-618-4
38-4111-9
1 37'2 I n -3}
|84-8±4-3
-o-2l8-o
8-61IO7
727110-;
+
-6711-5
i-5±4-o
-2-s±3-o
I75'9±5-I
}
}
PyBHK and BHK cells were harvested from confluent cultures by exposure either to EGTA
or EGTA + trypsin, as described in Materials and methods. These cells were then used to
measure the kinetics of cell attachment to plastic tissue culture dishes, films of denatured type I
collagen and 3-dimensional gels of native collagen fibres either in serum-free MEM or MEM
containing 5 % foetal calf serum. Data are presented as (a) the mean±s.D. of the percentage
of cells attached after 120 min of incubation, and (b) the difference between the percentage
of cells attached to each substratum in the absence of serum from that observed in the presence
of serum (a negative number therefore indicates that more cells attached in the absence of
serum than in the presence of serum). Data from 5 experiments are summarized in this table.
The results obtained in 3 experiments with subconfluent BHK cells are shown in
Table 2. In contrast to the results obtained with confluent cultures, subconfluent
BHK cells treated with either EGTA only or EGTA and trypsin show a significant
serum-dependence of attachment to films of denatured collagen. The attachment of
subconfluent BHK cells to gels of native collagen fibres is again not dependent on
serum. The results obtained with subconfluent cultures of PyBHK cells (data not
shown) are basically the same as shown in Table 1 for confluent cells.
276
S. L. Schor
Table 2. The attachment of BHK cells from subconfluent cultures
to collagen substrata
EGTA
Substratum
Plastic
Denatured
collagen films
Native
collagen gel
Serum % attachment
+
799 ± i89-S ± i37-8 ±3
86-9 ±286-2 ±i-
:}
EGTA + Trypsin
A attached
(+ serum) —
(— serum)
% attachment
— 9'6±2-2
{ 9 I-I±3-O}
-97±o-9
{%lll'l\
37-5 ±a-o
36-1 ±2-5
-4-3±3'i
A attached
(+ serum) —
(— serum)
f8i-8±o-9l
BHK cells were harvested from subconfluent cultures by exposure either to EGTA or
EGTA + trypsin. These cells were then used to measure the kinetics of cell attachment to
plastic tissue culture dishes, films of denatured type I collagen and 3-dimensional gels of native
collagen fibres either in serum-free MEM or MEM containing 5 % foetal calf serum.
DISCUSSION
Fibronectin is a major surface constituent of normal fibroblasts, but is absent from
the surface of their virally transformed counterparts (Pearlstein & Waterfield, 1974;
Hynes & Humphreys, 1974; Gahmberg, Kiehn & Hakomori, 1974; Hynes, 1976;
Vaheri & Mosher, 1978; Yamada & Olden, 1978). The morphological distribution
and quantity of fibronectin on cell surfaces varies as a function of cell density in
culture, with more fibronectin being associated with confluent fibroblasts than with
subconfluent ones (Pearlstein & Waterfield, 1974; Mautner & Hynes, 1977; Furcht,
Mosher & Wendelschafer-Crabb, 1978).
Fibronectin is also found in serum or plasma, where it is referred to as cold insoluble
globulin or CIG (Grinnell & Hays, 1978). Although the cell surface-derived and
serum-derived forms of fibronectin are immunologically indistinguishable and are
equally effective in mediating cell attachment to films of denatured collagen (Yamada
& Kennedy, 1979), important differences between these 2 forms of fibronectin have
been observed; these include differences in solubility, electrophoretic mobility and
ability to agglutinate red blood cells (Yamada & Kennedy, 1979; Yamada & Olden,
1978). Perhaps the most relevant difference between the cell surface-derived and
serum-derived forms of fibronectin in the context of the present discussion is the
reported inability of serum-derived fibronectin to bind directly to cell surfaces
(Pearlstein, 1978), whereas the cell surface-derived form will bind to cells and when
added to cultures of transformed fibroblasts results in a restoration of some of the
growth characteristics of normal cells (Yamada, Yamada & Paston, 1976). A considerable amount is known about the means by which serum-derived fibronectin
mediates cell attachment to films of denatured collagen. This form of fibronectin
binds avidly to collagen at a site between residues 757 and 791 on the ai(I) chain
Cell attachment to collagen
277
(Kleinman et al. 1978) and significantly more fibronectin binds to denatured collagen
than to native collagen fibres (Engvall & Rouslahti, 1977). As mentioned above, serumderived fibronectin does not bind directly to cell surfaces, and mediates cell attachment
to denatured collagen by first forming a complex with the collagen substratum,
followed by cell attachment to this fibronectin-collagen complex (Klebe, 1974).
Pearlstein (1978) has suggested that the interaction of fibronectin with collagen
induces a conformational change in the fibronectin molecule required for recognition
by receptors on the cell surface.
Cell surface fibronectin is extremely sensitive to trypsin and exposure of confluent
cultures of BHK cells to 10/ig/ml trypsin (50-fold less than used in this study) has
been observed to remove all detectable surface-associated fibronectin (Pearlstein &
Waterfield, 1974). On the other hand, cell surface fibronectin is not sensitive to
chelating agents such as EDTA and EGTA (Pearlstein & Waterfield, 1974). The
results reported in this communication indicating that confluent BHK cells removed
by exposure to EGTA only do not require serum for attachment to films of denatured
collagen, while such cells removed by EGTA and trypsin do require serum, are
consistent with the view that cell surface-associated fibronectin can mediate cell
attachment directly to denatured collagen. PyBHK cells and subconfluent BHK cells
either do not have surface-associated fibronectin or have an insufficient amount of it
and therefore require exogenous fibronectin to mediate cell attachment to denatured
collagen, regardless of whether or not trypsin is used to prepare the cell suspension.
In contrast, cell attachment to gels of native collagen fibres occurs by a fibronectinindependent mechanism (Schor & Court, 1979). This conclusion is supported by the
results presented here indicating that there are no differences in the kinetics of
attachment of both BHK and PyBHK cells following treatment with either EGTA
only or EGTA and trypsin to native fibres. These results also indicate that although
EGTA and trypsin remove different components of the cell surface, including
proteins and proteoglycans, none of these appear to influence the ability of cells to
attach to native fibres in the presence or absence of serum.
REFERENCES
L. J. & DERMIETZEL, P. (1976). Cell coat in tumour cells - effects of trypsin and
EDTA: A biochemical and morphological study. Oncology 33, 11-23.
BARD, J. B. L. & HAY, E. D. (1975). The behaviour of fibroblasts from the developing avian
cornea. J. Cell Biol. 67, 400-418.
BRUGMANS, M., CASSIMAN, J. J. & VAN DEN BERGHE, H. (1978). Selective adhesion and impaired
adhesive properties of transformed cells. J. Cell Set. 33, 121-132.
BuNCE, R. P. & BUNGE, M. B. (1978). Evidence that contact with connective tissue matrix
is required for normal interaction between schwann cells and nerve fibres. J. Cell Biol. 78,
943-950.
BURGER, M. M. (1969). A difference in the architecture of the surface membrane of normal and
virally transformed cells. Proc. natn. Acad. Sci. U.S.A. 62, 994-1001.
CASSIMAN, J. J. & BERNFIELD, M. R. (1974). Transformation-induced alteration in fibroblast
adhesion:masking by trypsin treatment. Expl Cell Res. 91, 31-35.
CODINCTON, J. F., SANFORD, B. H. & JEANLOZ, R. W. (1970). Glycoprotein coat of the TA 3
cell. (1) Removal of carbohydrate and protein material from viable cells. J. natn. Cancer Inn.
45. 637-647.
ANGHILERI,
278
S. L. Schor
T. & BARD, J. (1972). Collagen substrata for studies on cell behaviour. J. CellBiol. 54,
626-637.
ENGVALL, E. & ROUSLAHTI, E. (1977). Binding of soluble form of fibroblast surface protein,
fibronectin, to collagen. Int. J. Cancer 20, 1-5.
FURCHT, L. T., MOSHER, D. F. & WENDELSCHAFER-CRABB, G. (1978). Effects of cell density
and transformation on a fibronectin extracellular filamentous matrix on human fibroblasts.
Cancer Res. 38, 4618-4623.
GAHMBERG, C. G., KIEHN, D. & HAKOMORI, S. I. (1979). Changes in a surface labelled galactoprotein and in glycolipid concentrations in cells transformed by a temperature-sensitive
polyoma virus mutant. Nature, Lond. 248, 413-415.
GEY, G. O., SVOTELIS, M., FOARD, M. & BANG, F. B. (1974). Long-term growth of chicken
fibroblasts on a collagen substrata. Expl Cell Res. 84, 63-71.
GRINNELL, F. & HAYS, D. G. (1978). Cell adhesion and spreading factor. Expl Cell Res. 115,
ELSDALE,
221-229.
F. & MINTER, D. (1978). Attachment and spreading of baby hamster kidney cells
to collagen substrata: effects of cold insoluble globulin. Proc. natn. Acad. Set. U.S.A. 75,
4408-4412.
GROBSTEIN, C. (1953). Epithelio-mesenchymal specificity in the morphogenesis of mouse
8ub-mandibular rudiments in vitro. J. exp. Zool. 124, 383-414.
HODGES, G. M., LIVINGSTON, D. C. & FRANKS, L. M. (1973). The localization of trypsin in
cultured mammalian cells. J. Cell Sci. 12, 887-902.
HUET, C. & HERZBERG, M. (1973). Effects of enzymes and EDTA on ruthenium red and concanavalin A labelling of the cell surface. J. TJltrastruct. Res. 42, 186-199.
HYNKS, R. O. (1976). Cell surface proteins and malignant transformation. BiocJiim. biophys.
Acta 458, 73-107HYNES, R. O. & HUMPHREYS, K. C. (1974). Characterization of the external proteins of hamster
fibroblasts. J. Cell Biol. 63, 438-448.
KELLY, R. O. & LAUER, R. B. (1975). On the nature of the external surface of cultured human
embryo fibroblasts. Differentiation 3, 91—97.
KLEBE, R. J. (1974). Isolation of a collagen-dependent cell attachment factor. Nature, Lond.
GRINNELL,
250, 248-251.
KLEINMAN, H. K., MCGOODWIN, E. G., MARTIN, G. R., KLEBE, R. J., FIETZEK, P. P. &
WOOLLEY, D. E. (1978). Localization of the binding site for cell attachment in the ai(I)
chain of collagen. J. biol. Chem. 253, 5642-5646.
I. R. & HAUSCHKA, S. D. (1965). Cell and tissue interactions in the reproduction
of cell type. In Reproduction: Molecular, Subcellular and Cellular (ed. M. Locke), pp. 243-290.
New York: Academic Press.
KOSHER, R. S. & CHURCH, R. L. (1975). Stimulation of in vitro somite chondrogenesis by
procollagen. Nature, Lond. 258, 327-330.
LASH, J. W. & VARAN, N. S. (1977). Tissue interactions and extracellular matrix components.
In Cell and Tissue Interactions (ed. J. W. Lash & M. M. Burger), pp. 101-113. New York:
Raven Press.
LIOTTA, L. A., VEMBU, D., KLEINMAN, H. D., MARTIN, G. R. & BOONE, C. (1978). Collagen
required for proliferation of cultured connective tissue cells but not their transformed
counterparts. Nature, Lond. 272, 622-624.
MAUTNER, V. & HYNES, R. O. (1977). Surface distribution of LETS protein in relation to the
cytoskeleton of normal and transformed cells. J. Cell Biol. 75, 734-768.
MEIER, S. & HAY, E. D. (1974). Control of corneal differentiation by extracellular materials.
Collagen as a promotor and stabilizer of epithelial stroma production. Devi Biol. 38, 294KONIGSBERG,
270.
S. & HAY, E. D. (1975). Stimulation of corneal differentation by interaction between
cell surface and extracellular matrix. J. Cell Biol. 66, 275-291.
PEARLSTEIN, E. (1976). Plasma membrane glycoprotein which mediates adhesion of fibroblasts
to collagen. Nature, Lond. 262, 497-500.
PEARLSTEIN, E. (1978). Substrate activation of cell adhesion factor as a prerequisite for cell
attachment. Int. J. Cancer 22, 32-35.
MEIER,
Cell attachment to collagen
279
E. & WATERFIELD, M. D. (1974). Metabolic studies on 125 I-labelled baby hamster
kidney cell plasma membranes. Biochim. biophys. Ada 362, 1-12.
SCHOR, S. L. & COURT, J. (1979). Different mechanisms involved in the attachment of cells to
native and denatured collagen. J. Cell Set. 38, 267-281.
SNOW, C. & ALLEN, A. (1970). The release of radioactive nucleic acids and mucoproteins by
trypsin and ethylenediaminetetra-acetate treatment of baby hamster cells in tissue culture.
Biochem. J. 119, 707-714.
PEARLSTEIN,
STENN, K. S., MACKIE, J. A. & ROLL, F. J. (1979). Migrating epidermis produces AB2 collagen
and requires continual collagen synthesis for movement. Nature, Land. 277, 229-232.
A. & MOSHER, D. F. (1978). High molecular weight, cell surface-associated glycoprotein (fibronectin) lost in malignant transformation. Biochim. biophys. Ada 516, 1-25.
YAMADA, K. M. & KENNEDY, D. W. (1979). Fibroblast cellular and plasma fibronectins are
similar but not identical. J. Cell Biol. 80, 492-498.
YAMADA, K. M. & OLDEN, K. (1978). Fibronectins - adhesive glycoproteins of cell surface and
blood. Nature, Lond. 275, 179—184.
YAMADA, K. M., YAMADA, S. S. & PASTAN, I. (1976). Cell surface protein partially restores
morphology, adhesiveness and contact inhibition of movement to transformed fibroblasts.
Proc. natn. Acad. Sci. U.S.A. 73, 1217-1221.
VAHERI,
(Received 29 March 1979 —
Revised 26 May 1979)