Proc. Natl. Acad. Sci. USA
Vol. 75, No. 9, pp. 4408-4412, September 1978 Cell Biology
Attachment and spreading of baby hamster kidney cells to collagen substrata: Effects of cold-insoluble globulin (fibroblasts/fibronectin/connective tissue)
FREDERICK GRINNELL AND DIANNE MINTER Department of Cell Biology, The University of Texas Health Science Center at Dallas, Dallas, Texas 75235
Communicated by Jerome Gross, July 3, 1978
ABSTRACT Studies have been carried out to determine the effects of cold-insoluble globulin (CIG) on the attachment and spreading of baby hamster kidney cells on various collagen substrata. Cell attachment to native collagen substrata occurred in the absence of CIG just as fast as attachment to dried collagen or gelatin substrata occurred in the presence of CIG. On the other hand, cell attachment to dried collagen or gelatin was markedly reduced in the absence of CIG. Cell spreading also occurred on native collagen in the absence of CIG; however, CIG was absolutely required for cell spreading to occur on dried collagen or gelatin. Finally, anti-CIG antiserum or lactoperoxidase treatment inhibited cell spreading on CIG-coated substrata but not on native collagen substrata. The data are discussed in terms of the interaction of fibroblasts with collagen
shown that CIG has a much higher affinity for denatured collagen than for native collagen (21). Therefore, we have investigated the interaction of fibroblasts with native collagen gels to determine if CIG is required for this interaction to occur. METHODS AND MATERIALS Preparation of substrata All substrata were prepared in Falcon 3001 (35-mm) tissue culture dishes. The substrata were prepared by two general methods: gelation or adsorption. In the former instance, collagen solutions were added to the dishes and then polymerized, thereby forming a gel. In the latter instance, dishes were exposed to protein solutions for a short time and then extensively rinsed with deionized water. Protein adsorption to the substratum is known to occur almost instantaneously on exposure of the substratum to protein-containing solutions (22-24). With whole serum, the adsorbed layer of protein is about 20-50 A thick (5, 25). In general, pure protein solutions result in the formation of a monomolecular layer of adsorbed protein molecules that can be desorbed only under very harsh conditions (26). Dried Collagen Gels. This substratum has been used by Klebe (16) to demonstrate a serum protein required for cell attachment to collagen. Aliquots (1.0 ml) of a freshly prepared rat tail collagen solution, ca. 2 mg/ml in 0.1% HOAc, were gelled at 220 for 30 min in the presence of an NH3 atmosphere. The final pH was 11. Subsequently, the gels were air dried for 48 hrs at 220. Hydrated, Native Collagen Gels. The technique for preparing native collagen gels composed of cross-striated fibrils and their microscopic appearance were described by Gross and Kirk (19) and by Elsdale and Bard (20). Aliquots (1.0 ml) of a freshly prepared rat tail collagen solution, ca. 2 mg/ml in 0.1% HOAc, were brought to physiological ionic strength and pH at 40 by the addition of 10 times concentrated phosphate-buffered saline and NaOH and then placed at 370 for 30 min in a humidifiedchamber. The gels that formed were about 2 mm thick. These substrata were then used immediately. Gelatin-Coated Substrata. Aliquots (1.0 ml) of a freshly prepared rat tail collagen solution, ca. 2 mg/ml in 0.1% HOAc, were heated to 50° for 10 min. The solutions were then cooled to 370 and incubated in Falcon dishes for 10 min at 22°, following which the dishes were extensively rinsed with deionized
The interaction of fibroblasts with a substratum results in a series of biochemically discrete steps including contact of the cells to the substratum, formation of initial bonds of attachment, and reorganization of the cell cytoskeleton accompanied by formation of additional bonds of attachment leading to cell spreading (1). With a variety of cell lines including baby hamster kidney (BHK), HeLa, Chinese hamster ovary (CHO), and L, cell spreading under normal tissue culture conditions requires that the substratum surface be coated by a serum factor that has been identified as cold-insoluble globulin (CIG) (2, 3). The extent of cell spreading is related to the density of the factor adsorbed on the substratum surface (4). On the other hand, several cell strains, including W1-38, MRC-5, and human conjunctiva cells, have been shown to attach and spread in the absence of serum or CIG (5-7). Significantly, cell strains generally synthesize and secrete higher levels of the large external transformation-sensitive (LETS) protein [cell-surface protein (CSP), fibronectin], which is immunologically related to CIG, than cell lines or transformed cells (8-11). Moreover, in serum-free medium, cells secrete substances beneath themselves onto the substratum (5, 12-14). These observations have led to the hypothesis (15) that the ability of fibroblastic cells to spread in serum-free medium depends upon the extent to which the cells are able to secrete CIG or a CIG-like protein onto the substratum. In general, cell strains do not require the addition of exogenous CIG or serum, whereas CIG or serum is required for most cell lines and transformed cells. The adhesion of fibroblasts has also been studied with collagen substrata; however, in all of these studies, the investigators have used dried collagen gels prepared at high pH (16-18). This method of preparing collagen gels is quite drastic compared to usual techniques (19, 20) and might result in the collagen becoming denatured. Indeed, quantitative binding studies have
H20. Native Collagen-Coated Substrata. Aliquots (1.0 ml) of a freshly prepared rat tail collagen solution, ca. 2 mg/ml in 0.1% HOAc, were incubated in Falcon dishes for 10 min at 22°,
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Abbreviations: CIG, cold-insoluble globulin; ASF, fetal calf adhesion and spreading factor. 4408
Cell Biology: Grinnell and Minter following which the dishes were extensively rinsed with deionized H20.
Preparation of proteins Rat tail collagen was prepared according to the method of Price (27). CIG was prepared from human plasma by a modification of the techniques described by Mosher and Blout (28) and Mosher (29) that will be reported in detail elsewhere (3). The specific spreading activity of the CIG used in these experiments was 500-700 spreading units per mg of protein (see below). Assay of cell attachment and spreading The methods for measuring cell attachment and spreading of BHK cells have been described previously (2-4). To quantitate cell attachment, BHK cells (subline BHK-21-13s) were harvested from suspension cultures by centrifugation and washed and resuspended in adhesion medium (150 mM NaCl/3 mM KCl/1 mM CaCl2/0.5 mM MgCl2/6 mM Na2HPO4; pH 7.3). Incubations of ca. 0.75 X 106 cells in 1.0 ml of adhesion medium were placed in various collagen-treated Falcon culture dishes. The adhesion incubation assays were carried out for the time periods indicated at 370. CIG was added where designated. At the end of the incubations, the flasks were subjected to shaking at 150 rpm on a New Brunswick R-2 reciprocating shaker for 10 sec at room temperature, and the cells resuspended by this procedure (considered to be nonattached) were removed with a pipette. The turbidities of the starting and final cell suspensions were determined at 640 nm with a Bausch and Lomb Spectronic 70 equipped with digital readout. Cell concentrations were calculated from a previously determined linear relationship between cell number and turbidity. The percent 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. The precision and validity of this technique have been established. To determine cell spreading, BHK cells were suspended in adhesion medium as above, and incubations of ca. 0.5 X 106 cells in 1.0 ml of adhesion medium were placed in various collagen-treated Falcon culture dishes. The spreading assays were carried out for the times indicated at 370. CIG was added where designated. At the end of the incubations, the extent of cell spreading was determined visually with a Zeiss Invertoscope D inverted microscope equipped with phase contrast objectives and a Polaroid camera attachment. In instances when we wished to compare cell spreading, activity was determined by visually observing 100-200 attached cells and estimating the percentage of spread cells: 5-35% (1+), 40-60% (2+), 65-85% (3+), 90-100% (4+). With experience, it has become possible to read the qualitative assay with considerable reproducibility and little variation among three different observers. It should be pointed out that BHK cells have an absolute requirement for CIG in order to spread onto Falcon dishes, although they can attach to clean dishes nonphysiologically in medium with no serum (1). The effect of CIG on cell spreading on Falcon dishes is the same whether CIG is added to the incubation medium or used to pretreat the dishes for 5 min at 220 (2-4). One unit of spreading activity has been defined as the amount of serum protein or purified CIG required to promote complete spreading of BHK cells on Falcon dishes in a 45-min assay (2, 4). Spreading experiments were carried out with human fibroblasts similarly as described above, except that the cells were harvested from logarithmically growing stationary cultures by treatment with a trypsin/EDTA solution (Grand Island Biological Company, Grand Island, NY) for 10 min at 37°.
Proc. Natl. Acad. Sci. USA 75 (1978)
Cells BHK-21-13s cells were the gift of Adrian Chappel, Communicable Disease Center, Atlanta, GA. The cells were grown in suspension culture in Eagle's minimal essential medium (spinner modified) with double the concentrations of amino acids (except for standard glutamine) and vitamins and supplemented with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) buffer (20 mM), 0.1 g of ferric nitrate per liter, 2.0 g of dextrose per liter, 10% tryptose phosphate broth, and 10% fetal calf serum. The final sodium bicarbonate concentration in the medium was 0.5 g/liter and the pH was adjusted to 7.2. Human skin fibroblasts were the generous gift of Jim Griffin, The University of Texas Health Science Center at Dallas. The cells were grown in stationary culture in McCoy's 5A modified medium (Grand Island Biological Company) supplemented with Hepes buffer (20 mM, pH 7.2) and 10% fetal calf serum. Human fibroblast cultures were discarded prior to the 20th passage. RESULTS Time course of BHK cell attachment On dried collagen gels, attachment did not exceed 20% after 1 hr of incubation (Fig. 1). Addition of CIG to the medium resulted in a marked increase in the rate of attachment, and more than 80% of the cells were attached after 1 hr. This observation is similar to that made previously by others using dried collagen substrata (16-18). However, the conditions for gelation and subsequent drying of the gels are harsh treatments compared to the conditions known to result in formation of native collagen gels (20, 21). Therefore, it was possible that the dried collagen gels were denatured and had partially or completely lost their fibrillar organization. In support of this notion was the observation that dried collagen gels in contact with filter paper impregnated with 0.1% trypsin (Sigma type XI) were hydrolyzed following an overnight incubation at 37°. Hydrated, native collagen gels were unaffected by this treatment (data not shown). When hydrated, native collagen gels were tested for cell attachment (Fig. 1), there was no CIG dependence. That is, the rate of cell attachment to hydrated, native collagen gels was the same in the presence or absence of CIG in the medium and comparable to the rate of cell attachment to dried collagen gels in the presence of CIG. In order to test the possibility that CIG is required for cell 100
C) 80 60
00 15 30 45 60 0 15 30 45 '60 Minutes
FIG. 1. Time course of BHK cell attachment on various collagen substrata. CIG was either omitted from the incubation medium (U) or added at 10 units/ml (@). (A) Dried collagen gel; (B) native collagen gel; (C) gelatin coating; (D) collagen coating.
Cell Biology: Grinnell and Minter
adhesion to heat-denatured collagen, gelatin samples were prepared and used to coat the substratum. In control experiments, untreated collagen solutions were also used to pretreat the substratum. The results indicate that CIG is required for cell attachment to heat-denatured collagen-coated substrata but not to native collagen-coated substrata (Fig. 1). BHK cell spreading Experiments were carried out under conditions similar to those described in Fig. 1 in order to analyze cell spreading on the various collagen substrata. Cells remained rounded and no cell spreading was observed in the absence of CIG on dried collagen gels (Fig. 2) or gelatin-coated substrata (Fig. 3). The addition of CIG to the medium resulted in cell spreading (Figs. 2B, 3B, and 3C), and all the cells spread with CIG at concentrations >4 units/ml (Figs. 2B and 3C). Pretreatment of the denatured collagen substrata for 5 min at 220 with a CIG-containing solution also promoted subsequent spreading onto the substrata (Figs. 2C, 2D, and 3D); about 4-fold higher CIG concentrations were required for complete spreading to be observed (Figs. 2D and 3D). On native collagen-coated substrata, partial cell spreading occurred in the absence of CIG in 30 min (Fig. 4A), and spreading was complete by 2 hr (Fig. 4C). However, the addition of CIG to the incubation medium at 10-20 units/ml promoted the rate of cell spreading, which was best demonstrated after 30 min (compare Fig. 4 B with A). After 2 hr, cell morphology appeared the same in the presence or absence of CIG (Fig. 4 D and C). The effect of CIG at early times could also be observed after pretreatment of the native collagencoated substrata with CIG. On hydrated, native collagen gels, cell spreading was different from that observed on any of the other substrata. For one
FIG. 2. BHK cell spreading on dried collagen gels. The incubations were carried out for 45 min. (A) Control; (B) + CIG in the incubation medium at 5 units/ml; (C) substratum pretreated for 5 min at 220 with CIG at 5 units/ml; (D) substratum pretreated for 5 min at 220 with CIG at 20 units/ml. (X200.)
Proc. Natl. Acad. Sci. USA 75 (1978)
t FIG. 3. BHK cell spreading on gelatin-coated substrata. The incubations were carried out for 45 min. (A) Control; I(B) + CIG in the medium at 2 units/ml; (C) + CIG in the medium at 4 units/ml; (D) substratum pretreated for 5 min at 220 with CIG at 16 units/ml.
thing, the shape of spread cells was more bipolar and less triangular (compare Fig. 5 with Figs. 2-4; see ref. 19). Moreover, cell spreading never reached 100% even when incubations were carried out in complete growth medium overnight. Typically, about 25% of the cells were spread after 30 min (Fig. 5 A and
roG. 4. krHK cell spreading on native collagen-coatea substrata. The incubations were carried out for 30 min (A and B) or 2 hr (C and D). (A and C) Controls; (B and D) + CIG in the medium at 20 units/ ml. (X200.)
Cell Biology: Grinnell and Minter
FIG. 5. BHK cell spreading on hydrated native collagen gels. The incubations were carried out for 30 min (A and B) or 2 hr (C and D). (A and C) Controls; (B and D) + CIG in the medium at 20 units/ml. (X200.)
B) and 50-75% by 2 hr (Fig. 5 C and D), after which there was further change. The addition of CIG to the incubations had only a marginal effect on cell spreading even at the highest concentration tested, 20 units/ml. At most, there was a slight increase. (Compare Fig. 5 B with A and 5 D with C.) In experiments in which the hydrated native collagen gels were pretreated with CIG solutions, no effects were observed. Immunological and biochemical analysis of cell spreading The freshly prepared collagen utilized in these experiments was not purified; therefore, it was possible that CIG might be present as a contaminant in the preparations. This could explain the lack of a CIG requirement for cell attachment and spreading on native collagen substrata if such a CIG contaminant were lost or inactivated during preparation of the dried collagen gels or gelatin-coated substrata. However, sodium dodecyl sulfate gel electrophoretic analysis of the collagen preparations did not reveal a high molecular weight (210,000-250,000) polypeptide. Experiments were carried out to compare the immunological specificity of cell spreading on hydrated native collagen gels compared to CIG-coated dried collagen gels. Antisera prepared in rabbits against the fetal calf adhesion and spreading factor (anti-ASF, 4) and against human CIG (anti-CIG, 3) were found to crossreact with and inhibit the spreading activity-i.e., CIG-in rat serum. Clean culture dishes were pretreated with medium + 10% rat serum for 10 min at 220. BHK cell spreading on these dishes was found to be inhibited by the addition of the antisera to the spreading assays. The inhibition was 50% with anti-ASF (1:5 dilution) and 30% with anti-CIG (1:5 dilution) (averages of 4 determinations). Preimmune serum was without effect. It should be pointed out that the rat tail collagen preparations might contain rat CIG as a contaminant. Neither no
Proc. Natl. Acad. Sci. USA 75 (1978)
anti-ASF; nor anti-CIG was inhibitory toward cell spreading on hydrated native collagen gels, suggesting that rat CIG was not present as a contaminant. In marked contrast, anti-CIG (1:5 dilution) partially inhibited cell attachment and completely inhibited cell spreading on denatured collagen gels that had been treated with human CIG at 10 units/ml for 5 min at 220. We have shown elsewhere (3) that, under these conditions, the inhibitory effects of the antisera are directed at adsorbed CIG on the substratum and not on the cells. Finally, we have found that a brief, 10-min treatment of CIG adsorbed to a substratum with lactoperoxidase (0.01 mg/ml) and NaI (1.0 mM) in phosphate-buffered saline, pH 7.0, followed by 0.1 mM H202, resulted in an inhibition of the spreading activity of adsorbed CIG. These studies will be reported in detail elsewhere. Similar experiments were carried out with hydrated native collagen gels, and it was found that this treatment did not alter cell attachment or spreading onto the gels. Physiology of cell adhesion Despite the differences in CIG requirements for cell attachment and spreading on the various collagen substrata, the physiology of attachment and spreading was similar in every case. It has been previously shown that CIG-dependent cell attachment and spreading on tissue culture dishes or dried collagen gels can be inhibited by sulfhydryl binding reagents, energy metabolism inhibitors, or removal of divalent cations from the incubation medium (1, 30). Similar experiments were carried out with native collagen-coated substrata and native collagen gels, and a typical experiment is shown in Table 1. The results indicate that the addition of N-ethylmaleimide (0.1 mM) or 2,4-dinitrophenol (1 mM), or removal of divalent cations from the incubation medium inhibits cell attachment and spreading. Attachment and spreading of human fibroblasts All of the experiments carried out thus far were with BHK cells, an established cell line. In order to demonstrate CIG dependence of cell spreading with tissue culture dishes, it is necessary to use cells that do not secrete their own CIG-like factors (e.g., most cell lines). The hypothesis has been presented (15) that some cells (e.g., cell strains) may attach and spread on a layer of CIG or a CIG-like substance endogenously produced and elaborated by the cells. If this is correct, diploid human fibroblasts ought to attach and spread onto dried collagen substrata Table 1. Physiology of cell attachment and spreading* Native
Cell spreading Native
Control + MalNEt, 0.1 mM + MalNEt, 0.01 mM + MalNEt, 0.001 mM + DNP, 1 mM + DNP, 0.1 mM + DNP, 0.01 mM 0.16 M NaClt
2+ Ot 1+
2+ Ot 1+
2+ Ot 2+ 2+
2+ Ot 1+ 2+ Ot
Ot * N-Ethylmaleimide (MalNEt) or 2,4-dinitrophenol (DNP) was added to the incubations, as indicated. The extent of cell spreading was determined after 60 min (native collagen gels) or 45 min (native
collagen-coated substrata). extent of attachment was not quantitated in these experiments; however, it was obvious that no attachment at all occurred. Cells rinsed and resuspended in 0.16 M NaCl.
Cell Biology: Grinnell and Minter
i FIG. 6. Human fibroblast cell attachment and spreading. The incubations were carried out for 60 min on tissue culture plastic (A) or dried collagen gels (B and C). (C) + CIG in the incubation medium at 10 units/ml. (X200.)
in the absence of added CIG. Indeed, this is exactly what occurs, shown by the experiment shown in Fig. 6. Attachment and spreading occurred in the absence of serum or CIG on tissue as
culture substrata (Fig. 6A) or dried collagen gels (Fig. 6B), and there was little, if any, effect of adding CIG (10 units/ml) to the incubation medium (Fig. 6C). DISCUSSION This paper presents quantitative studies of cell attachment and spreading on hydrated native collagen gels. The findings demonstrate that BHK cells attach and spread on these substrata, which are composed of cross-striated fibrils, without the addition of CIG to the incubation medium. On the other hand, very little attachment and no spreading of cells occurs on dried collagen gels or gelatin-coated substrata unless CIG is added to the medium or used to pretreat the substrata. It is unlikely that the difference between adhesion to native collagen substrata and dried collagen or gelatin substrata can be accounted for by the presence of CIG as a contaminant in the collagen preparations. Antibodies directed against CIG do not inhibit cell spreading on native collagen gels but do inhibit cell attachment and spreading on CIG-coated denatured collagen substrata. Moreover, iodination of tyrosine residues inhibits the activity associated with adsorbed CIG gels but not native collagen gels. Nevertheless, the underlying physiology of adhesion with native collagen substrata is the same as that previously observed with CIG-coated tissue culture dishes (1) or dried collagen substrata (30)-i.e., a requirement for free SH groups, active energy metabolism, and divalent cations. The simplest interpretation of the data is that native collagen in the fibrillar form (hydrated, native gels) and microfibrillar form (native collagen-coated substrata) contains sites with which BHK cells can directly interact via appropriate cell surface receptors. These sites are apparently lost on dried collagen gels (partially or completely denatured fibrils) or substrata coated by gelatin (dispersed, randomized molecules), because BHK cells do not appear to interact directly with these substrata. Therefore, BHK cells may recognize collagen in some physical organizations but not others. Nevertheless, BHK cells are able to attach to CIG-coated dried collagen or gelatin substrata, suggesting that CIG acts as an adhesive bridge in these cases. These findings are consistent with the observation that CIG binds to gelatin much better than to native collagen (21). This
Proc. Natl. Acad. Sci. USA 75 (1978)
interpretation suggests that in situ fibroblasts may interact with native collagen fibrils directly and not via a CIG bridge. However, if there are regions in situ where collagen has become locally altered so as to permit CIG binding, cells probably attach to these regions via a CIG bridge. Finally, the experiments carried out with the human fibroblast cell strain are consistent with previous studies that have demonstrated cell strains to be able to attach and spread in the absence of serum or CIG (5-7), presumably by the elaboration of CIG or a CIG-like molecule (15). Note Added in Proof. Unlike BHK cells, CHO cells have been found to require CIG for attachment to native collagen gels. However, if there was phosphate present during collagen polymerization or in the incubation medium, CHO cells attached to but did not spread on native collagen in the absence of CIG (H. Kleinman, personal communication). On the other hand, we have found that BHK cells attach and spread on native collagen gels even if Hepes buffer is used in place of phosphate during collagen polymerization and subsequent incubations. We are indebted to Dr. Andrew Kang and Dr. Jerry'Gross for their helpful discussions during the course of this research and in preparation of the manuscript. This research was supported by a grant from the National Institutes of Health, CA14609. 1. Grinnell, F. (1976) in Membranes and Neoplasla: New Approaches and Strategies, ed. Marchesi V. T. (Alan R. Liss, New York), pp. 227-236.
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