J. Cell Sci. 19, 653-667 (1975) 653

Printed in Great Britain

TRYPSINIZED BHK21 CELLS AGGREGATE IN

THE PRESENCE OF METABOLIC INHIBITORS

AND IN THE ABSENCE OF DIVALENT

CATIONS

J. G. EDWARDS, JANICE A. CAMPBELL, R. T. ROBSONANDM. G. VICKER

Department oj Cell Biology, Tlie University, Glasgow Gn 6NU, Scotland

SUMMARY

The rapid formation of adhesions in suspension by lightly trypsinized BHK21 cells is notdependent on protein synthesis, and only in part on cellular metabolism, although it is com-pletely inhibited by heat- and aldehyde-fixation of the cells. A requirement for protein synthesisbecomes evident only if cells are exposed to high levels of trypsin for long periods. Formationof adhesions does not require addition to the medium of divalent cations, although it is increasedby divalent manganese and cobalt ions. It is promoted by cytochalasin B and by cyclic AMPand is not inhibited by £-mercuriphenylsulphonate. We discuss a possible relationship betweenaggregation and the formation of gap junctions.

INTRODUCTION

BHK21 cells (clone 13, Stoker & Macpherson, 1964) dispersed from culture ona substrate by trypsin and EDTA, adhere rapidly to one another in suspension(Edwards & Campbell, 1971). Cultured fibroblast-like cells also form intercellularadhesions when they encounter one another by locomotion on a substrate, adhesionswhich may be important in determining their social behaviour (Abercrombie, 1970)and which can generate a coherent cell sheet (James & Taylor, 1969). We do notknow whether cells in freshly prepared suspensions adhere to each other by the samemechanism as cells moving on a substrate. However, since in suspension locomotionis not required for cells to encounter one another, formation of adhesions in thismanner is more readily investigated (Gerisch, 1961; Moscona, 1961). We havepreviously shown that the rapid aggregation of clone 13 cells in suspension isenhanced by neuraminidase (Vicker & Edwards, 1972), inhibited by anti-tubulinalkaloids (Waddell, Robson & Edwards, 1974) and much reduced in some cell linesderived from clone 13 cells by transformation by polyoma virus (Edwards, Campbell& Williams, 1971).

The formation of intercellular adhesions may, in principle, depend on a complexsequence of cellular events involving various intracellular activities as vvell as the cellsurface (Edwards, 1975, for a review). To aid interpretation at the molecular level ofthe effects of these various agents on aggregation, we wished to know to what extentmetabolic activity by the cells is required for the formation of adhesions in suspension.

654 J- G. Edwards, J. A. Campbell, R. T. Robson and M. G. Vicker

Experiments which we now report show that aggregation of cells dispersed fromculture by brief exposure to trypsin is not dependent on protein synthesis, but is tosome extent dependent on glycolysis or electron transport. A requirement for proteinsynthesis becomes evident only if cells are treated with trypsin for longer periods.In the course of this work, we were surprized to find that formation of adhesions bythese cells in suspension is largely independent of added divalent cations.

MATERIALS AND METHODS

Cells

We discard cultures of BHK21 cells, clone 13, grown as previously described (Edwards &Campbell, 1971) after about a month, and recover fresh cells from a common frozen stock.Despite this precaution, we have encountered considerable variation in the extent to whichfreshly dispersed cells aggregate in the first hour from trypsinization. (See for example, Table1, p. 656.) One source of this variation is the culture density (Edwards & Campbell, 1971),another is overgrowth of cultures by a non-aggregating variant which has been cloned in thislaboratory by Mrs J. Dysart, and which we will describe elsewhere (in preparation).

In this report we refer to 2 different procedures for preparing cell suspensions, respectively'low' and 'high' trypsin. In the low-trypsin procedure (modified only slightly from that de-scribed by Edwards & Campbell, 1971) we drain trypsin-EDTA from the cultures as soon as ithas covered their surfaces, and resuspend the cells for aspiration in growth medium, rather thanTris-saline. After a first centrifugation, we resuspend the cells in the medium in which they areto be aggregated (Hanks' solution buffered with Hepes, 001 M, pH 7-4, except where otherwisespecified) and in the same medium after second centrifugation. For high-trypsin dispersal, wewash cultures twice with (Glasgow modified) Eagle's medium and overlay then with Eagle'smedium containing 200/tg/ml recrystallized trypsin and 10/tg/ml deoxyribonuclease I, 10 mlon 120 cm1 culture area. We incubate the cells for 15 min at 37 °C under a gas phase of aircontaining 5 % COa, aspirate to obtain a single cell suspension, and continue incubation for afurther 15 min. We then add calf serum to 10%, chill the suspension, recover the cells bycentrifugation at 5 °C and wash them once by resuspension in the medium to be used foraggregation.

Aggregation

We have measured aggregation by using a Coulter counter (Model Za) with a 2oo-/(maperture, to follow the reduction in total particle count from initial value iV0 to value at time t,N,, as described by Edwards & Campbell (1971) and discussed in detail by Edwards (1973).We incubate 4 0 ml cell suspension (io9 cells per ml) in 10-ml silicone-treated conical flasks, at37 °C in a reciprocating shaker, stroke 4 cm, 92 per min and dilute o-i-ml aliquots in 20 mlice-cold 09 % sodium chloride for counting.

Figs. 1, 2, and 4-6 show typical progress curves for the treatments described. In Tables 3and 4 we summarize data from a series of experiments with each agent, each determinationmade on a different day, usually with cells from a different passage level.Mean inhibition is

Nt inhibitor — Nt control . s-*oo5i/nlj — - — and 95 % confidence interval is — ,

JV0 control — N, control N/(n — 1)

where n is the number of observations and j the sample standard deviation.

Chemicals

Recrystallized trypsin, type III, 10-13000 BAEE units per ml, deoxyribonuclease I, 2000Kunitz units per ml, cycloheximide, rotenone, 2-deoxyglucose, dibutyryl-3',5'-cycIic adenosinemonophosphate and theophylline were all obtained from Sigma Chemical Company. Cyto-

Aggregation of trypsinized BHK21 cells 655chalasin B was from Aldrich Chemicals. Rotenone was administered as a solution in dimethylsulphoxide (British Drug Houses, spectroscopic grade) and cytochalasin B in o-i % dimethylsulphoxide, as described by Goldman (1972). Divalent cations were added as Analytical Gradechlorides.

RESULTS

Requirement for protein synthesis

We have prepared cell suspensions from cultures of BHK21 cells (clone 13) usingtwo different regimes of exposure to trypsin, to investigate the sensitivity of aggrega-tion of these suspensions to cycloheximide. The first regime, referred to as 'low

Fig. 1. Effect of cycloheximide on aggregation of BHK cells. Cells were dispersed bythe 'high' (# , O) or 'low' (A, A) trypsin procedures, and aggregated in completegrowth medium, with a gas phase of air/5 % CO2, in the presence (A, • ) or absence(A. O) °f cycloheximide, 10 /tg/ml. The ordinate N,/No shows the reduction in totalparticle count from initial value No to value at time t, Nt.

trypsin' (see Materials and Methods) is a standard procedure for dispersing cellsfor routine culture, and is designed to minimize the exposure needed to prepare amonodisperse cell suspension. This procedure is close to that used in all our previousstudies of the aggregation of trypsinized BHK cells. Cells are exposed to 0-5 mg perml Difco 1.250 trypsin for 4 min in the presence of 0-44 mM EDTA. In the secondregime (referred to as 'high trypsin') cells are exposed to 0-2 mg per ml recrystallizedtrypsin for 30 min in Eagle's medium. It is important to note that the specific activityof recrystallized trypsin is approximately 10 times that of Difco 1.250.

The aggregation of 'low trypsin' cells is completely insensitive to cycloheximide,whereas the conspicuously slower aggregation of 'high trypsin' cells is markedly,though not completely, inhibited by cycloheximide from its earliest stages (Fig. 1and Tables 1, 2). This comparison holds whether we aggregate the cells in a mediumcontaining glucose and salts only, or in complete growth medium. (We obtain differentresults if we expose cells to high concentrations of trypsin in the presence of EDTA.Rapid aggregation is then much more sensitive to trypsin, and slow aggregation occursonly after a lag which may be of several hours.)

656 J. G. Edwards, J. A. Campbell, R. T. Robson and M. G. Vicker

There is a striking contrast between the rapidity with which trypsin at low concen-tration disperses aggregates formed in suspension (less than io/ig/ml for 10 min(Edwards & Campbell, 1971)) and the extensive pre-treatment with trypsin requiredto convert rapid aggregation to the slow, cycloheximide-sensitive pattern. Indeed, inthe high-trypsin procedure, cells are freed from their substrate and readily dissociable

Table 1. Aggregation of low-trypsin cells is insensitive to 10 fig/mlcycloheximide

Experiment

i

2

345678

Hanks' HepesHanks' HepesHanks' HepesHanks' HepesEagle's, 250 /ig/mlEagle's, 250 /ig/mlGrowth mediumGrowth medium

Medium

, soy-bean trypsin inhibitor, soy-bean trypsin inhibitor

Control

O-2O

027

0-330-380-64

O"440-15

0-33

NJN0

Cycloheximide

020

0-25

o-35038067040

0-17

Table 2. Aggregation of high-trypsin cells is inhibited by 10 figjmlcyclolieximide

Experiment

1

2

3

4S67

Medium

Hanks' HepesHanks' HepesEagle's, 250 /ig/ml,soy-bean trypsin inhibitor

Growth mediumGrowth mediumGrowth mediumGrowth medium

Control

2-6 ± 0 1 2

2 2 ± O - l 8

2-9 ±054

3'5±°-i52-3 ±0073 - O ± O - I I

3-8±o-o6

Cycloheximide

1 -o ±0-25

0-7 ±o-ioo-8±O'i3

i ' 3±o i4o-6 ±022o-8±o-i6I-I ±030

% Inhibition

62

6773

6375747 i

Values are rates of aggregation ——I — I, x io3 min l, determined by least squares regression

of Ni/N0; values per experiment, 0—200 min; ±sample standard deviation from regressioncoefficient 6.

from one another by aspiration in less than 5 min, but abolition of rapid aggregationrequires a further 20-30 min exposure to the enzyme. It is therefore easy, by usingintermediate conditions, to obtain mixed kinetics in which there is some rapid cyclo-heximide-insensitive aggregation followed by a second phase of slower aggregationwhich is sensitive to this inhibitor (Fig. 2).

We can observe re-acquisition of adhesiveness by high trypsin cells in another way.If such cells are maintained for several hours suspended at low density in growthmedium, then recovered by centrifugation, washed and resuspended, they aggregaterapidly (Fig. 3). This procedure separates in time cellular activity dependent on pro-tein synthesis from the formation of adhesions, and also shows that the re-acquisition

Aggregation of trypsinized BHKzi cells 657

of adhesiveness is due to a change in the cells and not the medium. A similarcellular change presumably occurred in the experiments of Weiss & Maslow (1972),who found that maintenance in suspension decreased the sensitivity to cycloheximideof the aggregation of chick embryonic neural retina cells.

10

100Time, mln

Fig. 2

2C0

Fig. 2. Effect of cycloheximide on aggregation of BHK cells. Cells were dispersed asfor the 'high' trypsin procedure, except that trypsin was at an intermediate level(S° MS/ml, 15 min), and aggregated in complete growth medium, with a gas phase ofa' r/s % CO2, in the presence ( • ) , or absence (O) of cycloheximide, 10 fig/ml.Fig. 3. Recovery of rapid aggregation by ' high' trypsin cells. Cells dispersed by 'high'(A, O) o r 'low' (A, • ) trypsin (2 experiments) were maintained in suspension in asiliconized vessel with a Teflon-coated magnetically driven stirrer, in growth mediumequilibrated with air/5 % CO,, at a cell density 04 x io6/ml and at 37 °C. At timesshown on the abscissa, aliquots of cells were recovered by centrifugation, washed 3times by resuspension in a calcium- and magnesium-free Tris-buffered saline, andaggregated in Hanks' medium at a density of 05 x io8/ml for 1 h.

Requirement for cellular metabolism

Although cycloheximide does not inhibit the aggregation of low-trypsin cells,sodium fluoride does (Fig. 4 and Table 3) and so do respiratory inhibitors such ascyanide and rotenone (Table 3) provided glucose is omitted from the medium or 2-deoxyglucose is present simultaneously. (In other studies of early aggregation, bothOrr & Roseman (1969) and Daday (1972) found no inhibition of aggregation of chickembryonic neural retina cells by respiratory inhibitors, but did not test these agentsin the absence of glucose.) Glucose itself consistently promotes aggregation whenadded to cells suspended in glucose-free medium (Table 3).

Evidently some metabolism-dependent cellular activity, which can be driven byglycolysis alone, contributes to the formation of adhesions. Inhibition is however onlypartial, and somewhat variable between cell suspensions. We interpret this to meanthat the actual bonding event does not of itself require cellular metabolism. Thealternative explanation, that we fail to block completely the relevant metabolic

658 J. G. Edwards, J. A. Campbell, R. T. Robson and M. G. Vicker

pathway, seems very unlikely, since first: respiratory inhibitors are much more effectivein preventing the adhesion of identically pepared cells to serum [films (Robson &Edwards, in preparation); and secondly: pre-incubation with inhibitors such as cyanideor fluoride for 1 h does not increase the extent of inhibition. Aggregation is, however,completely inhibited at low temperature (Edwards & Campbell, 1971). A possibleexplanation is that even in the absence of metabolism, formation of adhesions istemperature-dependent because it requires passive lateral mobility (Frye & Edidin,1970) of a component in the cell surface.

0-4

20 40Time, min

Fig. 4. Effect of sodium fluoride on aggregation of BHK cells. 'Low ' trypsin cellswere aggregated in Hanks' Hepes medium, in the presence (A), or absence (O) of10 HIM sodium fluoride. # , sodium fluoride added at 15 min.

In the presence of glucose, aggregation can be slightly stimulated by the additionof L-glutamine (Fig. 5, Table 3). We examined this effect in detail to see if it indi-cated a requirement for hexosamine synthesis as described for other cell types byOppenheimer, Edidin, Orr & Roseman (1969) and by Oppenheimer (1973). Gluta-mine stimulation is abolished by the antagonists DON and Azaserine, but we werenever able to obtain stimulation of aggregation by hexosamines. Such negative evi-dence is not entirely compelling, but it seems most likely that glutamine stimulatesaggregation of these cells merely because it is a better energy source than glucose. Theantagonists could act by preventing entry of glutamine to cells or mitochondria,rather than by inhibiting synthesis of glucosamine phosphate. This clearly does notapply to the cells studied by Oppenheimer et al. (1969), which were stimulated toaggregate by hexosamines in the presence of glucose.

It is only possible to speculate what kinds of cellular activity linked to glycolysis orelectron transport could contribute to formation of adhesions in this assay. It seemsunlikely that membrane ruffling or extension related to cell locomotion is involved,since cytochalasin B at 5 /ig/ml, a concentration which completely inhibits membraneruffling in a few minutes in L cells (Carter, 1967), causes a distinct stimulation of

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660 J. G. Edwards, J. A. Campbell, R. T. Robson and M. G. Vicker

aggregation of lightly trypsinized cells (Table 3). (Interpretation of this result is,however, complicated by our recent finding that the concentration dependence ofeffects of cytochalasin B both on aggregation and attachment of cells to serum filmsis very sensitive to the concentration of dimethyl sulphoxide, used as a vehicle forthe cytochalasin, to which the cells are exposed (Hurum, Robson & Edwards, inpreparation).)

20 40

Time, min

Fig. 5

Fig. 5. Effect of L-glutamine on aggregation of BHK cells. 'Low' trypsin cells wereaggregated in Hanks' Hepes medium in the presence ( • ) , or absence (O) of 5 rnML-glutamine.Fig. 6. Effect of dbc AMP and theophylline on aggregation of BHK cells. Cells wereaggregated in Hanks' Hepes medium in the presence ( • ) or absence (O) of 1 mMdbc AMP and 01 mM theophylline.

Aggregation is also stimulated by dibutyryl-3',5'-cyclic adenosine monophos-phate (dbc AMP) in the presence of theophylline. This effect differs from the stimula-tion by neuraminidase which we described previously (Vicker & Edwards, 1972)and that by cytochalasin B in that it is largely an effect on the initial rate of aggregation,which is not apparent at longer times (Fig. 6, Table 3). Hsie & Puck (1971) found thatvarious morphological effects of the dbc AMP on CHO cells are reversed by agentsthat disrupt microtubules. Since we have shown that colchicine and vinblastine in-hibit aggregation of BHK21 cells (Waddell et al. 1974) it seems likely that the stimula-tion of aggregation by dbc AMP is mediated by tubulin. A similar conclusion wasreached by Shields & Pollock (1974) for the effect of dbc AMP in protecting BHK21cells from detachment from a substrate by EGTA. Aggregation is insensitive to5 x io~* M ^-mercuriphenylsulphonate, and so presumably does not depend on

Aggregation of trypsinized BHKz I cells

662 J. G. Edwards, J. A. Campbell, R. T. Robson and M. G. Vicker

sulphydryl groups exposed at the outside of the surface membrane. It is, however,completely inhibited by heat- or aldehyde-fixation (Table 3).

Lack of a requirement for divalent cations

Omission of divalent cations from the suspending medium inhibits aggregation oflow-trypsin cells very little (Table 4). Whilst we cannot exclude that a low concentra-tion of divalent ions leaking from the (washed) cells, or divalent cations tightly boundto the cell surface contribute to the formation of adhesions, this observation opera-tionally distinguishes the formation by these cells of adhesions to each other in sus-pension from their formation of adhesions to serum-coated substrates. This followsbecause adhesion to serum films of identically prepared cell suspensions at the samecell concentration is completely inhibited under those conditions of divalent cationdepletion which fail to inhibit aggregation (Robson & Edwards, in preparation).Aggregation is, however, inhibited by EDTA to a degree approaching the inhibitionby metabolic inhibitors (Table 4). That BHK21 cells really do form adhesions inEDTA has been confirmed many times by microscopic inspection. Inhibition byEDTA presumably results at least in part from interference with cellular metabolism,since EDTA is known to make BHK21 cells leaky (Snow & Allen, 1970). If the partialinhibition of aggregation by EDTA can be laid at the door of the subsidiary energy-linked activity, we can conclude that actual intercellular bonding does not requireappreciable concentrations of divalent cations. It is of interest, however, that thenon-physiological divalent cations cobalt and manganese, which we have found bestto support attachment of these cells to serum films, also markedly stimulate aggrega-tion (Table 4). Although there are several reports of the stimulation by manganese ofattachment of cells to substrates (Garvin, 1968; Rabinovitch & De Stefano, 1973)this effect on the formation of intercellular adhesions appears to be a new finding.

The divalent cation requirement for adhesion of cells to culture substrates is not atpresent understood. We suppose that an enzyme, normally activated by magnesium,is required for some kind of cellular activity (possibly spreading) which is essentialfor attachment to serum substrates and also stabilizes intercellular adhesions formedin suspension. Manganese ions cause little aggregation of derivatives of BHK21 cellswhich show low aggregation in Hanks' medium (Robson & Edwards, unpublished).

DISCUSSION

Requirement for protein synthesis

In earlier experiments (Edwards & Campbell, 1971) we showed that aggregates ofBHK21 cells formed in suspension can be rapidly dispersed by low concentrations oftrypsin. We interpret this to mean that protein extending outside the plasma mem-brane is required for the stability of these adhesions. Cycloheximide-sensitive aggre-gation shows that at least some of the protein required for adhesion is produced bythe cells, and is not derived from the serum of growth medium. Since after incuba-tion in growth medium, high-trypsin cells can be washed and will then aggregaterapidly, the protein must remain bound to the cells under these conditions. It is not

Aggregation oftrypsinized BHK21 cells 663

necessary to assume that a specific protein involved in adhesion is being replacedindividually when cells aggregate slowly after high trypsin treatment. Slow aggrega-tion may be limited by the rate of wholesale exposure of new cell surface. We suggestthat caution should be exercized in the interpretation of those experiments wheresynthesis of some particular cell-surface constituent has been shown to be required forcell aggregation. (For example protein (Moscona & Moscona, 1966) and hexosamine(Oppenheimer et al. 1969).) Synthesis of new cell surface must depend on synthesis ofmany components, such as phospholipid, protein and hexosamine. Since the synthesisof these is likely to be coordinated, it does not follow that a species required for in-corporation of new surface and therefore for aggregation is directly involved in inter-cellular adhesion. In the case of the adhesion of suspended BHK21 cells, the evidencefor protein involvement is the sensitivity of aggregates to trypsin, and not the cyclo-heximide-sensitivity of aggregation. Taken together, however, these experimentsdefine a protein or proteins probably associated with the outside surface of the cellswhich is required for the stability of adhesions, although they do not show that it actsdirectly as an intercellular ligand. Indeed Revel, Hoch & Ho (1974) have recentlysuggested that trypsin is effective in detaching cells from culture substrates primarilybecause it acts, presumably indirectly, on intracellular cytoskeletal elements and there-by causes the cells to round up. This seems less likely to apply to dissociation ofaggregates, since at early times, at least, cells appear very little spread on each other.In further discussion, therefore, we make the simpler assumption that this protein,referred to as Pa, is directly involved in adhesion.

Requirement for cellular metabolism

Clearly, many different cellular activities could require linkage to glycolysis orelectron transport to contribute to the formation of adhesions. One model is possiblewhich links the relative insensitivity of aggregation to pre-treatment with trypsin onthe one hand, with the metabolic and microtubule requirement (Waddell et al. 1974)on the other. This insensitivity could arise if cells replace surface-exposed Pa by activeextrusion from an intracellular pool shielded from trypsin by the plasma membrane.A mechanism such as this has already been suggested by Kemp, Lloyd & Cook(1973) to explain why trypsinized cells aggregate in the absence of protein synthesis.

Respiratory inhibitors, fluoride and antitubulin alkaloids all inhibit aggregationonly partially. They may, though need not, act against a common cellular activity.Antitubulin alkaloids could affect extrusion of Pa by blocking extrusion of vesiclesbearing Pa at their surface, or by altering the array of newly emergent Pa at the cellsurface (Fig. 7, model 1). Aggregation of BHK21 cells resembles a number of exo-cytotic processes in being stimulated by cytochalasin B and inhibited by colchicine(exocytosis reviewed by Allison, 1973), although as we discuss below, does notshare their requirement for external calcium ions. An alternative model supposesthat Pa is always located in the cell surface, but that formation of adhesions altersits state of integration with the membrane so as to increase its sensitivity to trypticdigestion. Energy and microtubule dependence could then result from active re-distribution of Pa in the plane of the surface, perhaps initiated by the cell dispersal

664 J- G. Edwards, J. A. Campbell, R. T. Robson and M. G. Vicker

procedure, and analogous with capping (Fig. 7, model 2). Ryan, Borysenko & Kar-novsky (1974) have shown that in the absence of cell locomotion, cell surface receptorsfor concanavalin A on human polymorphonuclear leucocytes are swept into patchesby a system which is energy-dependent and colchicine-sensitive, but not inhibitedby cytochalasin B. Concentration of Pa in one or a relatively few parts of the cell

Model 1 Model 2

'Low' trypsin dispersal

Formation of adhesivepatches

f~\

1 and 2. Requirementfor tubulin, ATP, cyclic AMP

Inhibited by low temperatureand fixation. No divalentcation requirement

Fig. 7. Possible mechanisms for energy, tubulin and protein dependence of aggrega-tion of BHK cells. The scale of junctions and supposed adhesive component (P.) arearbitrary.

surface, either before or after formation of an intercellular adhesion, could account forthe apparent non-adhesiveness of small aggregates of cells (Edwards, 1973).

Regardless of mechanism, it is clear that the formation of adhesions in suspensionis sensitive to a number of aspects of cell physiology. Thus agents or treatments whichaffect the formation of adhesions may act on subsidiary cellular activities rather thanon the actual bonding event. We have previously stressed that this makes it moredifficult to design experiments to investigate the latter (Edwards, 1973).

Aggregation oftrypsinized BHK21 cells 665

Lack of a requirement for divalent cations

Whilst it is usual to stress the importance of divalent cations for the formation andstability of intercellular adhesions, there are several precedents for divalent cationindependence. First, the adhesions characteristic, of aggregation phase Dictyostelium(Beug, Katz & Gerisch, 1973) unlike those of vegetative cells, form in EDTA. Second,Wasteson, Westermark, Lindahl & Ponten (1973) report that hyaluronic acid-dependent aggregation of feline lymphoma cells is not dependent on added divalentcations. Unlike such hyaluronic acid-dependent aggregation however, aggregation oftrypsinized BHK21 cells is completely inhibited at low temperature (Edwards &Campbell, 1971) and our own unpublished observations show that it is not inhibitedin the presence of 12-5 /^g/ml bovine testicular hyaluronidase. We conclude thereforethat hyaluronic acid-dependent aggregation and the aggregation we have studieddepend on qualitatively distinct adhesive mechanisms. Third, in an ultrastructuralstudy of rat heart tissue perfused with a calcium- and magnesium-free medium, Muir(1967) found that unlike various other junctional specializations, the gap junctionsremained intact. This instance may be particularly pertinent to BHK cells, since in afreeze-cleave study of these cells in culture Revel, Yee & Hudspeth (1971) found gapjunctions as the only junctional specialization, indeed the only areas where the sur-faces of apposed cells approached closer than 40 nm. Further evidence that gap junc-tions may not require divalent cations for their stability is the observation by Cox,Kraus, Balis & Dancis (1974) that depletion of divalents fails to inhibit passage of asmall molecule from BHK cells to Lesch-Nyhan fibroblasts in culture, since gapjunctions are believed to be involved in such transfer (Gilula, Reeves & Steinback, 1972).

Intercellular junctions and the initiation of adhesion

It is not clear what relationship, if any, exists between the experimental measure-ment of the formation of adhesions by suspended cells and information available fromultrastructural studies of intercellular junctions. One view is that the kind of adhesioninvolved in the initiation of a cell contact not only precedes in time the appearance ofjunctional specializations, but that they may depend on quite distinct molecularspecies. For example, Roth, McGuire & Roseman (1971) distinguish the 'earlier'adhesions showing specificity in their experiments and 'later' adhesions characterizedby desmosomes, tight junctions, etc. Sheffield & Moscona (1969) report a marked lack ofspecialized junctions in conventional thin sections made in the early phases of reaggre-gation of dissociated cells. It seems possible, however, that molecular species whichin existing contacts are components of already identified junctional specializations,may, both in vivo and in experimental systems, play a significant role in initiationof adhesions. Such species may be present at the free surfaces of moving or dis-sociated cells, and be able to interact with other cells at very early times, even thoughthey are not then sufficiently ordered to be recognized ultrastructurally. Orderingcould then follow as the result of interactions depending on co-operation of junctionalcomponents on both cells.

Besides independence of added divalent cations, there are other parallels between43 C E L 19

666 J. G. Edwards, J. A. Campbell, R. T. Robson and M. G. Vicker

adhesion of BHK21 cells in suspension and properties reported for gap junctions.For instance, Epstein & Sheridan (1974) have found that low-resistance junctions canform in the absence of protein synthesis and metabolic energy production. The forma-tion of gap junctions is also stimulated by cyclic AMP (J. Sheridan, personal com-munication). We therefore suggest that molecular apparatus responsible for themechanical stability of gap junctions could play a part in the formation of adhesionsbetween fibroblasts in suspension.

We thank Gordon Campbell and Andrew Hart for excellent technical assistance. This workis supported by the Cancer Research Campaign.

REFERENCES

ABERCROMBIE, M. (1970). Control mechanisms in cancer. Eur. J. Cancer 6, 7-13.ALLISON, A. C. (1973). The role of microfilaments and microtubules in cell movement, endo-

cytosis and exocytosis. Ciba Fdn Symp. no. 14 (ed. R. Porter & D. W. Fitzsimons), pp. 109-143.Amsterdam and New York: Associated Scientific Publishers.

BEUG, H., KATZ, F. E. & GERISCH, G. (1973). Dynamics of antigenic membrane sites relatingto cell aggregation in D. discoideum. J. Cell Biol. 56, 647-658.

CARTER, S. B. (1967). Effects of cytochalasins on mammalian cells. Nature, Lond. 213, 261-264.Cox, R. P., KRAUS, R., BALIS, M. E. & DANCIS, J. (1974). Metabolic co-operation in cell

culture: studies of the mechanisms of cell interaction. In Cell Communication (ed. R. P. Cox),pp. 67-96. New York: Wiley.

DADAY, H. (1972). The mechanism of aggregation of neural retina cells in vitro. Wilhelm RmixArch. EntuiMech. Org. 171, 244-255.

EDWARDS, J. G. (1973). Intercellular adhesion. In Netv Techniques in Biophysics and CellBiology (ed. R. Pain & B. J. Smith), p. 1. New York: Wiley.

EDWARDS, J. G. (1975). Cell adhesion. In Mammalian Cell Membranes, vol. 4 (ed. G. A.Jamieson & D. M. Robinson). London: Butterworth. (In Press.)

EDWARDS, J. G. & CAMPBELL, J. A. (1971). The aggregation of trypsinized BHK21 cells. J. CellSci. 8, 53-71.

EDWARDS, J. G., CAMPBELL, J. A. & WILLIAMS, J. F. (1971). Transformation by polyoma virusaffects adhesion of fibroblasts. Nature, New Biol. 231, 147-148.

EPSTEIN, M. & SHERIDAN, J. (1974). Formation of low-resistance junctions in the absence ofprotein synthesis and metabolic energy production. J. Cell Biol. 63, p. 95 a.

FRYE, L. D. & EDIDIN, M. (1970). The rapid intermixing of cell surface antigens after forma-tion of mouse-human heterokaryons. J. Cell Sci. 7, 319-336.

GARVIN, J. E. (1968). Effects of divalent cations on adhesiveness of rat polymorphonuclearneutrophils in vitro. J. cell. Physiol. 72, 197—212.

GERISCH, G. (1961). Zell Funktionen und Zellfunktionwechsel in der Entwicklung vonDictyostelium discoideum. V. Stadienspezifische Zellkontaktbildung und ihre quantitativeErfassung. Expl Cell Res. 25, 535-554.

GILULA, N. B., REEVES, O. R. & STEINBACK, A. (1972). Metabolic coupling, ionic coupling andcell contacts. Nature, Lond. 235, 262-265.

GOLDMAN, R. (1972). The effects of cytochalasin B on the microfilaments of baby hamsterkidney (BHK21) cells. J. Cell Biol. 52, 246-254.

HSIE, A. W. & PUCK, T. T. (1971). Morphological transformations of Chinese hamster cells bydibutyryl adenosine 3 ' : 5' monophosphate and testosterone. Proc. natn. Acad. Sci. U.S.A.68, 358-361.

JAMES, D. W. & TAYLOR, J. F. (1969). The stress developed by sheets of chick fibroblasts invitro. Expl Cell Res. 54, 107-110.

KEMP, R. B., LLOYD, C. W. & COOK, G. M. W. (1973). Glycoproteins in cell adhesion. Progr.Surf. Membrane Sci. 7, 271-318.

MOSCONA, A. A. (1961). Rotation mediated histogenetic aggregation of dissociated cells. ExplCell Res. 22, 455~475-

Aggregation of trypsinized BHK21 cells 667

MOSCONA, M. H. & MOSCONA, A. A. (1966). Inhibition of cell aggregation in vitro by puro-mycin. Expl Cell Res. 41, 703-706.

MUIR, A. R. (1967). The effects of divalent cations on the ultrastmcture of the perfused ratheart. J. Anat. 101, 239-261.

OPPENHEIMER, S. B. (1973). The utilisation of L-glutamine in intercellular adhesion in ascitestumour and embryonic cells. Expl Cell Res. 77, 175-182.

OPPENHEIMER, S. B., EDIDIN, M., ORR, C. W. & ROSEMAN, S. (1969). An L-glutamine require-ment for intercellular adhesion. Proc. natn. Acad. Sci. U.S.A. 63, 1395-1402.

ORR, C. W. & ROSEMAN, S. (1969). Intercellular adhesion. I. A quantitative assay for measuringthe rate of adhesion. J. Membrane Biol. 1, 100-124.

RABINOVITCH, M. & D E STEFANO, M. J. (1973). Manganese stimulates adhesion and spreadingof mouse sarcoma ascites cells. J. Cell Biol. 59, 165-176.

REVEL, J. P., HOCH, P. & Ho, D. (1974). Adhesion of culture cells to their substratum. ExplCell Res. 84, 207-218.

REVEL, J. P., YEE, A. G. & HUDSPETH, A. J. (1971). Gap junctions between electrotonicallycoupled cells in tissue culture and in brown fat. Proc. natn. Acad. Sci. U.S.A. 68, 2924—2927.

ROTH, S. A., MCGUIRE, E. J. & ROSEMAN, S. (1971). Evidence for cell-surface glycosyl trans-ferases. Their potential role in cellular recognition. J. Cell Biol. 51, 536—547.

RYAN, G. B., BORYSENKO, J. Z. & KARNOVSKY, M. J. (1974). Factors affecting the redistributionof surface-bound concanavalin Aon polymorphonuclear leukocytes. J. Cell Biol. 62, 351-365.

SHEFFIELD, J. B. & MOSCONA, A. A. (1969). Early stages in the reaggregation of embryonicchick neural retina cells. Expl Cell Res. 57, 462-466.

SHIELDS, R. & POLLOCK, K. (1974). The adhesion of BHK and PyBHK cells to the substratum.Cell 3, 31-38.

SNOW, C. & ALLEN, A. (1970). The release of radioactive nucleic acids and mucoproteins bytrypsin and ethylenediaminetetraacetate treatment of baby-hamster cells in tissue culture.Biochem. J. 119, 707-714.

STOKER, M. & MACPHERSON, I. (1964). Syrian hamster fibroblast cell line BHK 21 and itsderivatives. Nature, Lond. 203, 1355-1357.

VICKER, M. & EDWARDS, J. G. (1972). The effect of neuraminidase on the aggregation ofBHK21 cells and BHK21 cells transformed by polyoma virus. J. Cell Sci. 10, 759-768.

WADDELL, A. W., ROBSON, R. T. & EDWARDS, J. G. (1974). Colchicine and vinblastine inhibitfibroblast aggregation. Nature, Lond. 248, 239-241.

WASTESON, A., WESTERMARK, B., LINDAHL, U. & PONTEN, J. (1973). Aggregation of felinelymphoma cells by hyaluronic acid. Int. J. Cancer 12, 169-178.

WEISS, L. & MASLOW, D. E. (1972). Some effects of trypsin dissociation on the inhibition ofreaggregation among embryonic chicken neural retina cells by cycloheximide. Devi Biol. 29,482-485.

(Received 23 April 1975)

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