Cellular adhesion to collagen

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Prmted in Sweden Copyright @ 1978 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/78/l 162.0470$02.00/O

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Cellular adhesion to collagen THOMAS F. LINSENMAYER, EILEEN GIBNEY, BRYAN P. TOOLE and JEROME GROSS. The Developmental Biology Laboratory, Departments of Medicine and Anatomy, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA

BALB/3T3 cells were released from tissue culture plates with EGTA, and their rates of attachment to collagen gels polymerized on Millipore filters were measured. Cell attachment in serum-free medium was 20-50% of that which occurred in medium containing 10% fetal calf serum (FCS). Cell attachment to gels pretreated with FCS and assayed in serum-free medium was identical with that of reels in FCS-containing medium. Thus, it seems there-are two separate mechanisms of attachment to collagen; one involving direct attachment and a second mediated by a serum component(s) which binds to collagen.

Summary.

In developing systems, cells might interact with extracellular matrix through specific adhesion to collagen. For example, during cornea1 morphogenesis, mesenchymal cells migrate through a preformed, epithelially derived extracellular matrix [3, 5, 131composed of two different types of collagen [lo]. Subsequently, these cells differentiate as cornea1 fibroblasts. In the present studies we have investigated the rate of cell adhesion to matrices composed of purified collagen. We have found that there probably exist at least two mechanisms for such adhesion; one involving direct contact with the collagenous substratum, and a second mediated by a serum component(s) as suggested by Hauschka & White [4], Klebe [8], Pearlstein [12] and Kleinman et al. [9]. Materials

and Methods

BALB/3T3 cells, a gift of Dr R. Roblin, were cultured in 150mm Falcon tissue culture plates containing 30 ml of Eagle’s Minimal Essential Medium (MEM) supplemented with 10% fetal calf serum (FCS) and antibiotics (Gibco, Grand Island, N.Y.). Collagen was extracted from cleanly dissected rat tail tendoris in 0.5 M acetic acid for 48 h. The solution was clarified by centrifugation and the collagen preci-

Fig. 1. Abscissa: time (min); ordinate: cpmx lo-“; (a, b) 0- - -0, Millipore filter; A---A, collagen; (c) 0-s -0,

FCS; A---A, no FCS. Attachment of [3H]thymidine-labelled, EGTA-released cells to uncoated Millipore filters and collagencoated filters in (a) serum-free medium and (6) medium containing 10% FCS; (c) attachment of cells to collagen-coated filters in serum-free medium (no FCS) and medium containing 10% FCS. Duplicate samples are shown for each time point. pitated by addition of 10% NaCl. The precipitate was collected, redissolved in 0.4 ionic strength potassium phosphate buffer, and re-precipitated by dialysis against 0.01 M Na,HPO,. Two additional precipitations were performed out of 0.4 ionic strength potassium phosphate buffer, one by adding cold absolute ethanol to a final concentration of 17%, and the second by adding NaCl to a final concentration of 20%. The last precipitate was redissolved in 0.5 M HAc, dialyzed against 0.5 M HAc and stored at 4°C. Collagen solutions were polymerized on 13 mm diameter, T&on-free Millipgre &hers (MF, Millipore Corporation, HATFOO013) using two different procedures. (1) Acid collagen gels were produced by pipetting 1OOAaliauots of 0.1 M HAc collagen solution bko filt&s, and the collagen polymerized by exposure to NH,OH vapors; (2) heat gels were produced by dialyzing the collagen from 0.1 M HAc into 0.4 ionic strenath notassium ohosnhate buffer. oH 7.6. nioettinn lOOh-ali&ots of tie sdlution onto’ Millipore *filters, and gelling the collagen by incubation at 37°C. In both cases, the filters were then equilibrated with 3-4 changes of Ca2+, Mp;*+-free phosphate-buffered saline (CMF-PBS), followed by two ihanges of complete phosphate-buffered saline (PBS). Most experiments were done using both tvoes of eels. Millipore tilt&s or &agenGoated filters were removed from PBS and equilibrated for several hours with MEM buffered with 25 mM Hepes. In various experiments, the basic medium (MEM) was additionally supplemented with 10% FCS, or 1.25% Bovine

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The gels were fluorographed and the fluorograms scanned with a densitometer (E.C. Apparatus Corp.).

Results Attachment to collagen in the presence or absence of serum. Fig. 1a shows an experitime (mm); ordinate: cpmxlO-$ (a) O-O, Millipore filter; A---A, collagen; (b) O-O, FCS; A---A, BSA. Attachment of [3H]thymidine-labelled, EGTAreleased cells to (a) uncoated Millipore filters and collagen-coated filters in medium supplemented with 1.25% bovine serum albumin (BSA); (b) attachment of cells to collagen-coated filters in medium supplemented with either 10% FCS or 1.25% BSA.

Fig.

2. Abscissa:

Sehm Albumin (BSA) (Cohn fraction V, Sigma). When BSA was added to medium the pH was readjusted to 7.4 before use. Cells were labelled with either 1 or 0.1 @i/ml [3H]thymidine (New England Nuclear) for either 24 or 48 h, according to Gulp [l]. They were then washed with CMF-PBS and incubated at 37°C with 20 ml of 2X low3M EGTA for 20 min on a rotary shaker. Adhering cells were released by pipetting, collected by centrifugation and resuspended in the medium with vigorous pipetting to break up clumps. Aliquots (100 ~1) of the labelled cell suspension were pipetted onto Millipore filters or collagen-coated filters, and the cells were allowed to attach at room temperature for selected periods of time. Then each filter was dipped twice into three changes of phosphate-buffered saline (PBS) to wash off unadhered cells, and the filter was put into a scintillation vial containing 10 ml of scintillation fluid (Aquasol, New England Nuclear). This is a modification of cell adhesion assays published previously [ 1,4, IS]. In every experiment, an index of the total number of labelled cells available for attachment to each Millipore filter was obtained by pipetting 100 ~1 aliquots of cells onto control filters, allowing them to sit for 60 or 90 min, and then drawing the medium through the filter by touching the bottom against a Kim-wipe tissue. Since the cells were not washed off in this procedure, these filters, when assayed by scintillation counting, served as controls for the number of cells capable of attaching. To test for the effects of trypsin release, cells were detached from their plates by a IO-min exposure to 0.25% trypsin (Gibco) at 37°C and then collected by centrifugation, resuspended in the medium to be tested and then assayed as above. Purified collagen gels were assayed for the presence of non-collagenous proteins such as LETS [6, 7, 16, 171. Gels were iodinated with 9 using the lactoperoxidase method and the labelled proteins were separated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis on 6% slab gels [6].

ment in which cells were allowed to attach in serum-free medium to collagen gels polymerized on Millipore filters and to naked Millipore filters. The Millipore filter was found to have a surface to which cells readily adhered so, in many experiments, attachment to Millipore filters was monitored as a control. In these experiments, within 3045 min after plating, the number of labelled cells adhering to the untreated filters after washing off the unattached cells plateaued at between N-80% of the total cells pipetted onto the filter. In serum-free medium, the rate of attachment to collagen gels, when compared to the naked Millipore filters, was slower, and only 20-50 % as many cells attached to collagen as did to the uncoated filters (fig. 1a, see also fig. 2a). When the assay was performed in medium containing 10% FCS, both the rate of cell attachment and the total number of cells attached to collagen were increased to equal that of the Millipore filter controls (compare fig. 1a to 1b). Fig. 1c describes an experiment in which cell attachment to collagen-coated filters was assayed in either serum-free medium or medium containing 10% FCS. To test whether the effect of serum in the medium was solely due to the presence of protein, which, for example could produce a “wetting effect”, we supplemented medium with BSA. Fig. 2a, b show the results of this supplementation. Fig. 2a compares collagen gels with naked Millipore filters when assayed in BSA supplemented medium, and fig. 2b compares the attachment to collagen gels in media supplemented Exp Cell Res 116 (1978)

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Fig. 3. Abscissa: time (mm); ordinate: cpmx 10e3. Attachment of [3H]thymidine-labelled, EGTAreleased cells in serum-free medium to uncoated Millipore filters (O-O) and collagen-coated filters (A-A) that had been previously treated with FCS.

with either FCS or BSA. It is evident that BSA does not mimic the effect of whole FCS, and yet again, data from a number of experiments show that 20-50% of the cells attach to collagen in the absence of FCS

during the 60-W min time period of the experiments. To determine whether the collagen in the substratum had been altered by binding some component from serum, we pretreated the gels with FCS, extensively washed the gels, and assayed cell attachment in serum-free medium. Fig. 3 shows cell attachment in serum-free medium to serum-pretreated collagen gels compared to naked Millipore filters. The attachment to collagen gels is now like that noted in serum-containing medium (compare fig. 3 with fig. 1b, c). Thus, it seems that some component(s) from serum binds to the collagen substratum and promotes cellular adhesion, over and above that which occtfrs to collagen itself. To test for the presence of LETS protein bound to the untreated collagen, polymerized gels were iodinated and the labelled

Fig. 4. Fluorograms with accompanying densitometric tracings of iodinated proteins separated on 6 % SDS gels. (Top) Untreated collagen gels; (bottom) collagen gels pretreated with LETScontaining plasma. Symbols designate the migration positions of al and a2 chains plus p chain dimers composed of two (~1chains (PI, J or one (Y1 and one a2 chain (&, J. 0, origin; J front.

EXP Cell Re., 116 (1978)

Preliminary 80

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BSA control gels and, as expected, fewer cells attached than in the presence of FCS (fig. 5). DISCUSSION

Previous studies have demonstrated that cells can attach to gelatinized collagen via an intermediary, non-collagenous protein Fig. 5. Abscissa: time (min); ordinate: cpmxlO-*; O-O, EGTA-FCS; O---O, trypsin-FCS; A-A, contained in serum [4, 8, 9, 121. Pearlstein EGTA-BSA, A---A, trypsin-BSA. Attachment of [3H]thymidine-labelled cells in me- [12] has shown that the serum protein is dium supplemented with either 10% FCS or 1.25% most probably the cell surface glycoprotein BSA. The cells had been released from the tissue culLETS [6, 7, 14, 16, 171. In the present ture plate by either EGTA or trypsin treatment. study, using a different cell line (BALB/ 3T3), a different method of releasing cells proteins were separated by electrophoresis (EGTA) and a different attachment assay, on 6% SDS slab gels. Fluorograms of un- we have confirmed that serum contains a treated gels and their densitometric scans component(s) capable of binding to collagen (fig. 4, top) show distinct bands correspond- and thus enhancing cell attachment. ing to collagen al and (r2 chains, p chain We have also noted that 20-50% of the dimers, and very high molecular weight ma- cells capable of attaching to the collagen terial near the origin which probably repre- gels in serum-containing medium will attach sents crosslinked multimeric forms of col- in serum-free medium. Our untreated gels lagen. Most importantly there is no labelled do not contain detectable levels of LETS material in the region where LETS is protein either by iodination as shown in the known to migrate (i.e. with a molecular present investigation or by fluorescent antiweight of 220 000, slightly slower than &, 1 body staining (data not shown). After inchain dimers) [ 16, 171.When equivalent col- cubation in serum-containing plasma, howlagen gels were treated with LETS-containever, our gels contained readily detectable ing plasma before iodination (fig. 4, bot- levels of LETS by both criteria. Also, since tom), fluorograms showed a dense band in lactoperoxidase catalyzed iodination labels the region where LETS protein migrates. the tyrosine residues in protein, one would To examine whether an intact cell surface expect this assay procedure to be heavily is required to effect binding to collagen, we weighted for the detection of LETS. Puriexamined the rate of cell adhesion of tryp- lied collagen contains only two residues of sin-released cells, which have many more tyrosine per 1000 amino acids whereas of their cell surface proteins removed, as LETS protein contains approx. 40 tyrocompared with EGTA-released cells [2]. In sines per 1000 amino acids [ 161. all cases the trypsin-released cells adhered In addition, the binding to collagen in more slowly than those released with serum-free medium is probably not due to EGTA and in most cases they plateaued in trivial reasons such as exposed areas of FCS-containing medium at about the same Millipore filters. The amount of collagen level as the EGTA-released ones (fig. 5). deposited is enough to leave a macroThese cells also attached more slowly to the scopically visible layer covering the surface

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of the filter, and in the scanning electron microscope this layer is composed of uniform, dense layers of tibrils. Both Klebe [8] and Pearlstein [12] also noted that cells could bind to non-serum treated collagen gels, and that this binding could be removed by treatment of their gels with 8 M urea at room temperature. They attributed this result to the removal of endogenous serum-binding factor in their collagen preparations. Maroudas [ 111, however, on theoretical grounds has raised the possibility that the 8 M urea treatment could partially denature the collagen gels, altering their fibrillar structure and thus masking any cell attachment mechanisms that might recognize native collagen tibrils. Our data strongly support this interpretation that there may indeed be at least two types of cellular binding to collagen; one directly to the native molecules or fibrils and one involving a serum-protein intermediate. We thank Dr Richard Hynes for performing assays for LETS protein. This is publication no. 747 of the Robert W. Lovett Memorial Group for the Study of Diseases Causing Deformities. Supported by grants from the NIH (EY02261, AM3564 and DE04220). Dr Linsenmayer is a recipient of the Research Career Development Award from the NIH (AMOO031) and Dr Toole is an Established Investigator of the American Heart Association (no. 73 138).

References

5. 6. 7. 8. 9. IO. Il. 12.

Culp, L A, J cell bio163 (1974) 71. Gulp, L A & Black, P H, Biochemistry 11 (1972) 2161. Dodson, J W & Hay, E D, Exp cell res 65 (1971) 215. Hauschka, S D &White, N K, Research in muscle develonment and the muscle spindle. Excerpta medica international congress series (1972) 63. Hay, E D & Revel, J P, Fine structure of the avian cornea (eh A Wolsky & P S Chen) vol. I. S Karger, Basel. Hynes, R 0, Proc natl acad sci US 70 (1973) 3170. Hynes, R 0 & Bye, J M, Cell 3 (1974) 113. Kiebe, R J, Nature 250 (1974) 248. Kleinman, H K, McGoodwin, E B & Klebe, R J, Biochem biophys res commun 72 (1976) 426. Linsenmayer, T F, Smith, G N & Hay, E D, Proc natl acad sci US 74 (1977) 39. Maroudas, N G, Nature 267 (1977) 183. Pearlstein, E, Nature 262 (1976) 497.

13. Trelstad, R L & Columbre, A J, J cell biol 50 (1971) 840. 14. Vaheri, A & Ruoslahti, E, Int j cancer 13 (1974) 579. 15. Walther, B T, Ohman, R & Roseman, S, Proc natl acad sci US 70 (1973) 1569. 16. Yamada, K M, Schlesinger, D H, Kennedy, D W & Pastan, I, Biochemistry 16 (1977) 5553. 17. Yamada, K M &Weston, J A, Cell 5 (1975) 75. Received April 17, 1978 Revised version received July 21, 1978 Accepted July 26, 1978

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Colchicine binding of cell extracts from colchicine-resistant mutants of Chlamydomonas reinhardi D. J. FLANAGAN of Biology, University YOI 5DD, UK

and J. R. WARR, Department of York, Heslington,

York,

The colchicine-binding activity of a high speed supematant from fourteen colchicine- and/or vinblastine-resistant mutants of Chlamydomonas reinhardi has been compared to that of wild type. Four of the mutants have reduced binding per unit protein. The low level of binding of one of these mutants is unusually stable. Three other mutants have normal initial binding levels, but show altered kinetics of decay of binding activity. Most of the mutants with altered colchicine-binding activitv oroduce abnormally large cells. Seven other mutants showed only slight or no differences in colchicine binding from wild type. Summary.

Several single gene mutations in Chlamyconfer changes in the levels of resistance to the antimicrotubular agents, colchicine and vinblastine [l-3]. Colchicine binding should provide an experimental approach to study the basis of resistance in these mutants, but a problem arises with the low level of the colchicinebinding activity in Chlamydomonas cell extracts. Plant material has a very low level of colchicine-binding activity in general [4], and only labile colchicine-binding activity can be detected in Chlamydomonas extracts [5]. It has not been proved that bind-

domonas reinhardi