Acquisition of synchronous beating between embryonic heart cell aggregates and layers

Experimental

ACQUISITION EMBRYONIC

Cell Research 113 (1978) 263-272

OF SYNCHRONOUS HEART

CELL

BEATING

AGGREGATES

BETWEEN

AND LAYERS

EVA B. GRIEPP and MERTON R. BERNFIELD Department

of Pediatrics. Stanford University School of Medicine, Stunford, CA 94305, USA

SUMMARY Synchronous beating between chick embryonic heart cell aggregates and heart cell layers was used to study the relationship between intercellular adhesion and ionic coupling. Adhesion was measured by counting the proportion of aggregates which were not to be removed from cell layers by gentle washing after a 30 min incubation. Synchrony between bound aggregates and contiguous layers was assessed by phase microscopy. The first evidence of synchrony was seen 1.5 h after addition of aggregates to layers. following which there was an increase in the percentage of aggregates beating synchronously, reaching over 50% at 7 h and slowly increasing to a maximum of 65% by 24 h. Scanning electron microscopy and autoradiography of thymidine-labeled cells suggest that synchrony does not depend on cell movement at the interface between aggregate and layer. Acquisition of synchrony can be prevented completely by inhibiting protein synthesis, although pulsation of aggregates and layers continues in proportions unchanged from controls. After reversal of protein synthesis inhibition, synchrony is acquired at a rate and to an extent closely resembling that of newly adherent controls. These data indicate that ionic coupling is neither an inevitable nor an immediate consequence of adhesion. Since ionic coupling has been shown to correlate with the presence of gap junctions, the findings suggest that gap junctions are not involved in the initial events responsible for intercellular adhesion in vitro and that their formation following adhesion in this system may depend upon protein synthesis.

In the hope of understanding better the role which cell surface phenomena play in bringing about the unique morphology characteristic of particular organs, we have investigated the relationship between intercellular adhesion and ionic coupling. Previous attempts to study how adhesion and coupling might be related have rarely distinguished between cell contact and specific cellular adhesion, which can be defined as contact which is selective, enduring and offers resistance to mechanical separation. Cell contact is obviously a prerequisite for coupling to occur [183, but specific cellular adhesion may also be required. The most suggestive data in this regard comes from 18-781812

Loewenstein’s study of marine sponges [22], in which coupling cannot be brought about by contact in the absence of a species-specific aggregation factor. Other workers have shown that coupling occurs rapidly after contact [6, 16, 22, 24, 301, but have not differentiated between contact and adhesion. Loewenstein’s observations and the rapid occurrence of coupling after contact have given rise to the hypothesis that coupling may play a role in specific adhesion. In a study of gap junction formation, Johnson et al. [ 181suggest that gap junctions may be intimately involved in adhesive recognition because the areas in which gap junctions

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paper [ 131, the system offers us the possibility of following the appearance and distribution of gap junctions by freeze-fracture techniques, an undertaking which would be extremely difficult with single cell pairs, and simplifies electrophysiological investigation of these small cells. 0 Our investigations of synchronization be0 1 2 3 4 6 6 7 8 9 10 "v-24 tween beating embryonic heart cell aggreFig. 1. Abscissa: time (hours); ordina&: synchronous aggregates. gates and layers following adhesion suggest Acquisition of synchronous beating between aggregate and contiguous layer. Following removal of non- that ionic coupling is not part of the proadherent aggregates, conditioned medium was added cess of adhesive recognition. In addition, and synchrony was evaluated. Time is from addition of aggregates to layers. The ordinate represents the we have evidence to indicate that protein synthesis may be required to initiate ionic bound aggregates which are beating synchronously. Each point represents the mean f SE. of at least coupling between heart cells. Preliminary four experiments, each involving a minimum of 15 reports of this work have previously apaggregates. peared [14, 151. 100

C

form are the areas of closest membrane apMATERIALS AND METHODS position. We have approached the question of Preparation of aggregate and layers hearts from seven-day-old chick embryos were whether ionic coupling is involved in ad- Whole removed and placed in a solution of 67% potassiumhesive recognition by measuring the bind- free Earle’s balanced salt solution (KF-EBSS) and fetal calf serum (FCS, Gibco). The hearts were ing of embryonic chick heart cell aggregates 33% minced, the fragments washed three times in calcium to heart cell layers under conditions which and magnesium-free EBSS containing 1.3 mm KC1 (CMF-EBSS) and incubated 10 min in this solution. allow these cell groups to beat, and have Crystalline trypsin (Worthington, 0.5 % in CMF-EBSS) considered the presence of synchronous was added to the heart cell fragments, which were then at 37°C for 30 min. Following trypsinizapulsation of aggregate and layer an indica- incubated tion, the heart fragments were rinsed with CMFtion of ionic coupling between them [13]. EBSS and were then mechanically dissociated in conmedium containing 20 pg/ml deoxyribonuWe feel that this system has certain advan- ditioned clease (DNase, Worthington) and 4% chick embryo tages over single cell systems although it extract (nreviouslv dialvsed aeainst KF-EBSS). The conditio;ed medium was prepayed by incubating a meexacerbates the uncertainty with regard to dium containina 70% KF-EBSS. 20% medium 199. cell type [27] which is present in any dis- 4% horse serum, 2 % FCS, penicillin (100 U/ml) and streptomycin (50 pg/ml [5]) with a low density of sociated heart cell preparation, and makes myocardial cells for four days [19], then filtered and it impossible to examine in detail the inter- buffered with 15 mM TES and 15 mM Hepes and until use. After dissociation, the cells were pelactions between individual cells. Since we frozen leted in a clinical centrifuge at 1500 g for 5-10 mitt, can easily wash off and count unbound resuspended in fresh conditioned medium, and filtered Nytex mesh with a pore size of 43 pm. Cell aggregates we can distinguish between ad- through layers were made by adding 2.5~ lo6 cells to each of hesion [4, 231and contact; we are also able several 1.8 cm2 wells of a multiwell tissue culture dish or Costar). Aggregates were prepared by placto study adhesion and coupling quantita- (Linbro ing 3X lo6 cells in 3 ml of conditioned medium plus tively in a system in which tissue-specific 20 pg/ml DNase in a 35 mm bacteriologic plastic Petri dish on a gyratory shaker at 84 rpm. Both layers and differences in adhesion can be measured aggregates were incubated for 24 h in humidified air [4]. As documented in the accompanying at 37°C.

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Adhesion method Aggregates and layers were washed twice with assay medium (80% KF-EBSS, 20% medium 199, buffered with 15 mM TES-HEPES). Aggregates of fairly uniform size (with a mean long axis of 28513 tim) were separated from debris, single cells and small aggregates by sedimentation at 1 g in a plastic centrifuge tube [4]. The aggregates and layers were incubated separately in assay medium at 37°C for 30 min following which 0.5 ml of a suspension of aggregates was added to the cell layer. After a 30 min incubation without shaking, the adhesion reaction was terminated by removing the medium containing the nonadherent aggregates and gently washing the layers twice with 0.5 ml warm assay medium. The nonadherent aggregates were pooled in an empty well. The aggregates remaining bound and those that were removed were counted under a dissecting microscope, and the ratio of bound to total aggregates was calculated. Details of the method, including the specificity and kinetics of aggregate binding, are described in Cassiman & Bernfield [4].

9 10 "18-24

Fig. 2. Abscissa: time (hours); ordinate: % of bound aggregates. Comparison of synchrony with other beating patterns observed. Time is from addition of aggregates to layers. The dotted line in each panel is from fig. 1 and shows the percentage of bound aggregates beatina synchronously ing svnchronouslv with the contiguous continuous (A) Aggregate and layer both-beatboth beatlayer. la;er.*(A) ina. ing, but asvnchronouslv. asynchronously. (B) Onlv Only laver layer beating. (Cj (C) Only aggregate ‘beating. beating. (0) Neither aggregate nor layer beating. Each point represents the mean f SE. of at least four experiments, each involving a minimum of 15 aggregates.

For determinations of rate, the pulsations of each aggregate or layer were counted for I min. Control aggregates were added to wells in which no layers were present. Comparisons were made with Student’s t-test.

Autoradiography Layers were prepared on 35 mm tissue culture dishes, and labeled with 5 &i/ml of f3H]thvmidine (suet. act. 2X lo3 /.&i/mmdle) for 3 h. Seven hours’after addition of aggregates to layers, the dishes were fixed in 2.5% glutaraldehyde in EBSS overnight and then dehydrated in alcohol. Layers and bound aggregates were removed from the dishes with amyl acetate [26] and then embedded in Epon. Sections l-2 pm thick were cut from six aggregate-layer pairs. The preparations were coated with NTB, emulsion and exposed for 4 weeks. They were developed in D19 for 6 min and stained with Toluidine Blue.

Scanning electron microscopy Assay for synchrony Conditioned medium was added to the aggregates which remained bound to the cell layers, and then both the aggregates and the layers contiguous to them were observed under phase microscopy at 100x, while the temperature was maintained at 37°C with an air curtain incubator (Sage). Each aggregate-layer was classified in one of five categories: (1) aggregate and layer beating synchronously; (2) aggregate and layer beating asynchronously; (3) aggregate only beating; (4) layer only beating; (5) neither aggregate nor layer beating. The percentage of aggregate-layers exhibiting each beating pattern (in ah-experiments in which more than half of all aggregates and contiguous layers were beating) was averaeed at half hourlv and hourlv intervals. PGsive motion of an aggregate-on a pulsaiinn layer could readily be distinguished from active be&i& of the aggregate, which involves a centripetal motion. If only questionable or partial synchrony of the aggregate with the surrounding layer was evident, the situation was scored as asynchronous.

Samples were prepared on 35 mm tissue culture dishes. Assessments for synchrony were carried out between 6 and 8 h after addition of aggregates, and the beating pattern of each aggregate was marked on a photograph of the dish. The entire dish was fixed with 2.5% glutaraldehyde in EBSS overnight, stained with I % osmium tetroxide in EBSS for 1 h on ice and then dehydrated in alcohol. The dishes were cut into pieces under alcohol and then dried above the critical point with Freon 13 or CO,. They were coated with gold palladium and observed in an ETEC scanning microscope. The morphology of each aggregate was assessed by two observers (either from a photograph independently or at the microscope concurrently) without knowledge of whether or not it was synchronous.

Transmission

electron microscopy

Aggregates were fixed in 2.5 % glutaraldehyde in EBSS (pH 7.4) overnight, followed by 1% osmium tetroxide

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Table 1. Effect of aggregate-layer

interaction

Aggregates on plastic

Aggregate Layer

No layer

Only aggregate beating

85.3k3.1 (48) Plastic

81.4k2.8 (32)

on pulsation

rate

Aggregates on layers Only layer beating

Asynchronous

Synchronous

6;.5+3.0 (22)

76.0+2. I (24) 69.Ok3.0 (24)

60.42 I .7 (33) 60.4+ I .7 (33)

Each number represents beats/min (mean f SE.), assessed 7 h after addition of aggregates to layers. Numbers in parentheses indicate number of observations. For comparisons of asynchronous with synchronous aggregates, pO.OS; when only one component is beating, comparison of aggregate with layer givesp<0.05.

in EBSS for 1 h at 4°C. Following 3 rinses with 0.2 N sodium maleate (pH 5.15), samples were stained with I % uranyl acetate in 0.2 N sodium maleate (pH 6) prior to dehydration in alcohol. They were then embedded in BEEM capsules in an Epon-Araldite mixture. After sectioning, samples were placed on Formvar-coated grids, and stained with 0.1% lead citrate.

Inhibition

of protein synthesis

Inhibitors of protein synthesis were added at the conclusion of the adhesion assay (30 min after addition of aggregates to layers). Both cycloheximide (Sigma) and puromycin (diHC1, Sigma) were used at final concentration of 10 pg/ml. In experiments involving reversal of inhibition, the drug was washed out by five rinses with conditioned medium. Controls for the assessment of recovery from cycloheximide consisted of untreated aggregates added to untreated layers 30 min prior to the washing procedure to remove cycloheximide from treated aggregates and layers, and are referred to as newly adherent controls. Protein synthesis was estimated by the incorporation of [‘Qeucine (New England Nuclear Corp.; 338 mCi/mmole; 0.5 FCi/ml) during a 3 or 6 h interval ending at 7 or 24 h following initiation of the adhesion reaction. Cultures were solubihzed in 3% Na dodecyl sulfate and the total protein estimated spectrophotometrically [19]. The sample was made 10% in trichloracetic acid, boiled for 5 min and cooled on ice for 45 min prior to collecting the precipitate on glass fiber titters. Filters were counted in a Beckman LS233 scintillation counter and the [W]leucine incorporation per mg protein calculated.

RESULTS Adhesion

Adhesion was assayed by determining the proportion of aggregates which could not be removed from cell layers by gentle washing 30 min after they were added; 86% re-

mained bound, a percentage in good agreement with the results from extensive studies of adhesion of quiescent heart cell aggregates and layers by Cassiman & Bernfield [4]. An equal proportion of bound and free aggregates were beating after termination of the adhesion reaction and during the first 7 h of observation, indicating that aggregate pulsation has no significant effect on adhesion. Acquisition

of synchrony

The gradual acquisition of synchrony between aggregates and the layers to which they are bound illustrated in fig. 1; the percentage of aggregates beating synchronously with the layer is plotted as a function of the time after the aggregates were added. A negligible proportion of aggregates was synchronous with the layer during the initial 1.5 h, following which there was a gradual increase in synchrony reaching 50% at 7 h. Sixty-five percent synchrony was achieved by 24 h. As the proportion of synchronous aggregate-layers increased, there was a concomitant decrease in the percentage of aggregate-layers beating asynchronously (fig. 2A). Some contribution to the increasing proportion of synchronously beating aggregate-layers was also made by a decrease in

Acquisition

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beating

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100

80

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60 Af

60 PROTEIN 40

SYNTHESIS

I% OF CONTROLI

40

t

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10 11 12 13 14 16 16 17 18-24

time (hours); ordinate: % synchronous aggregates. Acquisition of synchrony in presence of protein synthesis inhibitors. Incubation with inhibitors and control as in fig. 3. Each point represents the mean LSE. of at least four experiments.

Fig. 4. Abscissa: Fig. 3. Abscissa:

time (hours); ordinate:

% beating

aggregates. Beating of bound aggregates in presence of protein synthesis (% of controls) inhibitors. Time is from addition of aggregates to layers. Following removal of non-adherent aggregates, conditioned medium containing IO pg/ml (0-O) cycloheximide or (O--O) puromycin was added (A-A). Control incubations were run simultaneously and handled identically, but contained no drugs. Beating was considered present if either layer or aggregate or both were beating. Numbers in parentheses represent the percent of control values of [Wlleucine incorporation per mg protein during 3 or 6 h terminating at the time indicated. Each value is the average of at least two experiments.

Although changes in the pulsation rate of specific aggregates and layers with time was not followed, the proportions of aggregatelayers exhibiting different types of beating behavior imply that synchronization involves chiefly conversion of asynchronous situations (in which aggregate and layer the percentage of quiescent aggregates on are already beating). The apparent acquisibeating layers (fig. 2B). The proportion of tion of synchrony between pulsating layers situations in which the layer was not beatand inactive aggregates (but not vice versa) ing did not change substantially with time and the fall in pulsation rate with synchrony (fig. 2c, D). both suggest that the presence of a beating Physiological characteristics of layer may influence the aggregate with reaggregate-layers spect to initiation and rate of pulsation. The pulsation rates of aggregates and layers (Pulsation rate has previously been noted to are given in table 1. Aggregates beat at vary inversely with the volume of aggreessentially the same rate when bound to in- gates rather than depending upon a paceactive layers or to plastic. Aggregates seem maker [33]; the dramatic fall in the pulsato have a faster intrinsic pulsation rate than tion rate of aggregates with synchrony and layers, as can be seen from the data com- apparent induction of beating in aggregates paring the beating of aggregates on inactive by layers may reflect the impact of the layers to the beating of layers under inac- larger mass of coupled cells in the layer tive aggregates. When both aggregates and on the relatively smaller volume of coupled layers are beating asynchronously, their cells in the aggregate.) pulsation rates are not statistically different from one another. There is a significant de- Morphology of aggregates on layers crease both in aggregate and in layer pulsa- To try to clarify whether movement of cells tion rate when synchronous beating is at the interface between aggregate and layer achieved; the difference is more marked for might be involved in acquisition of synchrony, we labeled layers with [3H]thymithe aggregate than for the layer.

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Fig. 5. Abscissa: time (hours); ordinate: % synchronous aggregates. Acquisition Of synchrony following removal of cycloheximide. Incubation with cycloheximide and control as in fig. 3. At 6f h, fresh aggregates were added to a set of control layers so that removal of unbound aggregates in this control coincided with washing some of the cycloheximide-treated layers with fresh conditioned medium at 7 h (arrow). Each Doint reuresents the mean of at least two exoeriments.

dine and then examined by autoradiography the distribution of labeled cells in aggregatelayers 7 h after addition of aggregates to layers. No labeled cells were seen within aggregates. When examined in the scanning electron microscope, about half the aggregates were seen to be flattened, while the remainder retained more closely their original spherical configuration (fig. 6a, b). No correlation between the morphological appearance of the aggregates and synchrony was apparent when a total of 54 synchronous and asynchronous aggregate-layers were compared 7 h after addition of aggregates. That equal numbers of spherical and flattened aggregates beat synchronously suggests that spreading of cells from the aggregate onto the layer is not required for synchrony to occur. Cellular

composition

of aggregates

The extent to which sorting out of different cell types occurs within aggregates [27,

331was assessed since the presence of nonmyocytic cells in the interface between aggregate and layer might contribute to a delay in onset of synchrony. Transmission electron microscopic examination of thin sections of aggregates shows that only a few of the cells at the periphery of the aggregates contain myofibrils (as has previously been documented by others [20, 28]), suggesting that many peripheral cells may be of epicardial or endocardial origin. &“feect of inhibition

on acquisition

of protein

synthesis

of synchrony

We considered the possibility that the delay in acquisition of synchrony following adhesion occurs because time is required for synthesis of gap junctions precursors. Since proteins are known to be a major component of gap junctions, we decided to assess the effect of inhibitors of protein synthesis on the process of synchronization. Initial studies showed that effective inhibition of protein synthesis does not interfere with beating. Addition of 10 pglml cycloheximide to the medium at the conclusion of the adhesion assay allows vigorous beating of aggregates and layers for as long as 24 h in proportions comparable to controls, despite marked inhibition of [ 14C]leucine incorporation (fig. 3). Puromycin is an equally potent inhibitor of protein synthesis and also allows a high proportion of aggregates to beat for the first 7 h of observation. Although beating continues unabated in the presence of inhibitors of protein synthesis, the development of synchrony between aggregate and layer is completely prevented by the addition of 10 pg per ml of either cycloheximide or puromycin to the medium (fig. 4). Prevention of acquisition of synchrony by cycloheximide can be entirely reversed

Acquisition of synchronous beating

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Fig. 6. Morphology of aggregates on layers. Samples were assessed for synchrony 7 h after addition of aggregates to layers, then fixed and prepared for scan-

ning electron microscopy. (a) Shows a typical flattened aggregate (x440) and (b) a typical spherical aggregate (x380).

by removing the inhibitor medium and washing with medium free of drug; subsequently synchrony develops after a similar lag period, at a rate, and to an extent closely paralleling the acquisition of synchrony by newly adherent aggregate-layers (fig. 5). After the washing procedure, [‘“Clleucine incorporation in the formerly inhibited culture returns to 60-90 % of control values by 7 h. Aggregates are not removed from the layer by washing, suggesting that failure to attain synchrony in cycloheximide-treated cultures is not a consequence of loss of adhesion. Thus, inhibition of protein synthesis reversibly prevents acquisition of synchrony between aggregates and layers without affecting beating or adhesion.

bryo heart cells aggregates and layers suggests that functional integration between cells can develop following the formation of intercellular bonds in vitro, and encourages the belief that adhesion in vitro may be a phenomenon of relevance to in vivo embryogenesis. The method used in this study to assess adhesion has previously been shown to demonstrate differences in binding between aggregates and layers from various tissues [4] and can therefore be said to measure adhesive recognition. Since all aggregates are in contact with the layer, but only some remain bound to the layer after washing, the assay differentiates between contact and adhesion. In order to justify using our findings regarding synchrony to speculate on the possible role of gap junctions in adhesive recognition, we must first examine the experimental evidence that synchrony requires ionic coupling and the presence of gap junctions. Studies of myocardial cells from chick embryos [6], from rats [16], and from mice [ 111, have demonstrated

DISCUSSION An understanding of adhesive recognition and ionic coupling is vital to comprehension of how organs develop from individual cells. The occurrence of synchronous beating following adhesion between chick em-

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ionic coupling between contacting single cells which are beating synchronously with one another; cells within a synchronously beating aggregate of chick embryo heart cells have also been shown to be coupled [33]. In addition, a correlation has been reported between synchrony and the presence of gap junctions [6, lo] which are thought on the basis of fine-structural and electrophysiological observations to be the membrane components responsible for ionic coupling in diverse other systems [I, 2, 3, 7, 18, 25, 29, 31, 321. If synchrony is a reasonably sensitive index of the presence of gap junctions, as suggested by the accompanying paper [ 131, then the delayed and gradual acquisition of synchrony following adhesion, and the failure of some adherent aggregates ever to achieve synchrony both suggest that gap junctions are not required initially for formation of intercellular bonds in embryonic myocardial cells. The failure to remove asynchronous aggregates from layers after cycloheximide treatment despite extensive washing also suggests that gap junctions may not be involved in maintaining adhesion. The question of what causes the delay in acquisition of synchrony remains. The simplest explanation is that the increased lag between contact and coupling seen when our results are compared with the observations on single myocardial cells [6] involves the multicellularity of our system: even if all the cells at the interface were of myocardial origin, a few cells with delayed development of coupling might retard attainment of synchrony of the whole aggregate-layer [6]. The absence of a relationship between surface spreading of cells and synchrony, while suggestive, does not rule out the possibility that movement between aggregate

and layer cells at the interface between them is important in allowing synchrony to occur. The autoradiographic studies are more conclusive, although it is still possible to imagine, despite the evidence that the labeled layer cells do not enter the aggregate, that unlabeled cells from the aggregate move on the surface of the layer. Although we cannot absolutely exclude cell movement as a cause for the delay in acquisition of synchrony, the positive evidence that synchrony requires protein synthesis is not explained by a hypothesis requiring cell movement since there are data suggesting that cycloheximide does not interfere with cell movement [37]. Another possible cause for the delay in acquisition of synchrony in this multicellular system involves the presence of nonmyocytic cells at the periphery of the aggregates, and consequently at the interface between aggregate and layer. Since nonmyocytic cells are also on the periphery of aggregates which show specific adhesion, and since it has been established that cardiac cells are capable of achieving synchrony when coupled via other cell types [9, 10, II, 121, the presence of non-myocytic cells on the periphery of the aggregates may not be relevant. Nevertheless, it is conceivable that the kinetics of heterologous coupling might be different from the time course of coupling between homologous cells. Although we cannot exclude the possibility that the non-myocytic cells somehow insulate the aggregates from the layer, it is hard to imagine how inhibiting protein synthesis would in that case prevent acquisition of synchrony. We feel that it is most likely that the delay in acquisition of synchrony is related to formation of gap junctions in at least some cells at the interface between aggregate and layer. This hypothesis is supported by

Acquisition of synchronous beating the complete inhibition of acquisition of synchrony by cycloheximide and puromytin and the reversibility of the cycloheximide effect coincident with a resumption of protein synthesis. The similarity of the time course of acquisition of synchrony after reversal of cycloheximide to that seen in newly adherent controls suggests that protein synthesis is involved early in the process which leads to synchrony. Our data contradicts previous suggestions [35] that protein synthesis is not required for gap junction formation or the establishment of synchrony in heart cell cultures. This may well be due to differences in the experimental systems, which are not described in sufficient detail [35] for us to evaluate. In Novikoff hepatoma cells, cycloheximide does not prevent the formation of low resistance junctions [8]. This discrepancy could be explained by the fact that the hepatoma cells are not dissociated in trypsin which degrades junctional proteins (Finbow, Johnson & Revel, unpublished). Although we have not proven that synchrony is determined by formation of gap junctions rather than, for example, by degradation of some extracellular material between aggregate and layer, this possibility is greatly strengthened by freeze-fracture and electrophysiological studies which show that there are significant differences in various parameters of gap junctions and ionic coupling between adherent synchronous and asynchronous aggregate-layers [ 131. Synchrony between heart cell aggregates and layers is therefore a potentially useful assay for studying formation of gap junctions, and for investigating the relationship between gap junction structure and function. We wish to thank Dr Jean-Paul Revel for many stimulating discussions as well as for invaluable assistance with the electron microscope. We are also

27 1

grateful to Drs Paul Letourneau, Peter Ray and R. Lane Smith for their helpful suggestions, and to Paige Patch and Marjorie Weesner for preparation of the manuscript. This work was supported by a grant from the National Foundation-March of Dimes and NIH grant HD06763. E. B. G. is the recipient of NIH fellowship HD05076.

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