J. Cell Set. 40, 245-256 (1979) 245Printed in Great Britain © Company of Biologiiti Limited 1979

EFFECT OF ISOLATED PLASMA MEMBRANES

ON CELL COHESION IN THE CELLULAR

SLIME MOULD

ALIDA R. JAFFfi AND DAVID R. GARRODCRC Medical Oncology Unit, Centre Block, Southampton General Hospital,Southampton SOg 4XY, England

SUMMARY

The effects of isolated plasma membranes on cohesion of Dictyostelium discoideum Ax-2 cellshave been studied. Membranes isolated from cells in the log phase of growth gave completeinhibition of log-phase cell cohesion. This effect was specific for the cohesion of log-phase cellsmediated by contact sites B, since the cohesion of aggregation-competent cells which haveacquired contact sites A was only partially inhibited by log-phase plasma membranes. Mem-branes isolated from stationary phase cells gave partial inhibition of log-phase cell cohesion,while membranes from aggregation-competent cells gave complete inhibition of log-phase cellcohesion but at much higher concentration than log membranes. Treatment of log-phase cellswith cycloheximide for 8 h rendered them completely non-cohesive. Membranes from cyclo-heximide-treated cells had no effect on log-phase cell cohesion. Log-phase membranes gavecomplete inhibition of cohesion of 4 slime mould species. The results are disqussed in termsof our ligand-receptor model of log-phase cell cohesion.

INTRODUCTION

Immunological studies on the cell surface properties of D. discoideum cells haveprovided evidence for the existence of 2 independent cohesive mechanisms, contactsites A (CS A) and contact sites B (CS B), on the surface of aggregation stage cells(Beug et al. 1970; Beug, Katz & Gerisch, 1973).

Contact sites A appear at the time of aggregation, are insensitive to EDTA and areresponsible for the end-to-end cohesion of cells in aggregation streams. Contact sitesB which are present on vegetative cells and persist through aggregation, are involvedin side-to-side cohesion at this stage and are EDTA-sensitive. Both end-to-end andside-by-side cohesions have been shown to be completely blocked by univalent anti-body fragments (Fab) directed against membrane antigens of aggregation-competentcells (Beug et al. 1970).

Miiller & Gerisch (1978) have isolated a specific membrane constituent whichbehaves as the target site of adhesion-blocking Fab in aggregating D. discoideum cells.They have identified it as a Concanavalin A-binding glycoprotein with a molecularweight of 80000 Daltons. On the other hand, CS B of vegetative cells have not yetbeen characterized, and less is known about their nature and function.

On the basis of our previous work (Swan & Garrod, 1975; Swan, Garrod & Morris,1977; Garrod, Swan, Nicol & Forman, 1978; Jarre", Swan & Garrod, 1979) we have

246 A. R. Jaffi and D. R. Garrod

proposed a ligand-receptor model for vegetative cell cohesion, i.e. that mediated byCS B, of axenic D. discoideum cells. Our model suggests that both ligand and receptormolecules occur on the surface of log-phase cells. If this is correct, we would expectplasma membranes isolated from log-phase cells to bind to the surface of intact log-phase cells and inhibit their cohesion. Merrell & Glaser (1973), and Gottlieb, Merrell& Glaser (1974), have isolated plasma membrane-enriched fractions from chickembryonic neural retina and optic tectum. These fractions were capable of inter-acting preferentially with the homologous cells, preventing cell cohesion. Subsequentlythey have extracted glycoprotein components from the isolated plasma membranes,which inhibited cohesion of homologous cells (Merell, Gottlieb & Glaser, 1975).

Following a similar approach, we are attempting to isolate the molecular componentsof the cohesion system of vegetative slime mould cells. As a first step, we have isolatedplasma membranes from axenic log-phase cells, and shown that these do have aspecific inhibitory activity on log cell cohesion. We also extended these studies toshow lack of species-specific cohesion in that D. discoideum plasma membranes in-hibit the cohesion of 4 species, as would be predicted from the interspecific cohesionstudies by Nicol & Garrod (1978). It has been reported previously that partiallypurified plasma membranes inhibit development of D. discoideum (Smart & Tuchman,1976).

MATERIAL AND METHODS

Growth conditions

Axenic strain (Ax-2) cells of D. discoideum were grown as described previously (JaiK, Swan& Garrod, 1979). Log-phase cells were harvested when at a density of 2-5 x io* cells/ml;stationary phase cells were harvested when the cell count had remained constant at approxi-mately 1-2 x io7 cells/ml for 3-5 days. After harvesting, the cells were washed twice with colddistilled water before use.

Agar plate cultures

Cells of the species Polysphondylium violaceum (Pv), D. purpureum (Dp), D. mucuroides (Dm)and D. discoideum (Dd, NC-4) were grown as described by Swan et al. (1977) and harvested atfeeding stage, 36 h after inoculation of spores.

Acquisition of aggregation competence

Aggregation-competent cells were obtained as described previously (JafK et al. 1979) anddissociated with distilled water before use in cohesion assays or membrane isolation.

Cycloheximide treatment

Cells harvested at log phase of growth were suspended at 1 x io'/ml, in 17 mM phosphatebuffer, pH 6-o, containing 500 fig/ml of cycloheximide, and shaken at 140 rev/min at 22 °Cin a New Brunswick G-86 rotary shaker. The cells were washed twice in cold distilled waterbefore use in cohesion assays or plasma membrane isolation.

Cohesion assays

Cohesion was assessed as described previously (Jarfe et al. 1979). To test the effect of isolatedplasma membranes on cell cohesion, the cells were shaken in the presence of different con-

Plasma membranes and cell cohesion 247

centrations of plasma membranes, concentrations being expressed in terms of fig of membraneprotein per ml, and the cohesion assessed in the usual way. In all cases, the final concentrationof cells was 1 x io'/ml in 2 ml of 17 mM phosphate buffer, pH 6-o.

Plasma membrane isolation

After harvesting, the cells were washed twice in 17 mM phosphate buffer, pH 6-o, and oncein 30 mM Tricine buffer, pH 7-0. The cells were then resuspended in Tricine buffer at 1 x ioa/ml.An equal volume of a solution containing Tricine buffered-digitonin (1 mg digitonin/ml, inTricine buffer) was added to the cell suspension and the mixture stirred for 1 min at roomtemperature (Riedel & Gerisch, 1968). 60% sucrose was added to a final concentration of6 %, and the suspension stirred for another 15 min. This suspension was examined in the lightmicroscope to ensure that the majority of cells were lysed and cell ghosts were present.

After 30 min centrifugation at 13000 g (4 CC) the pellet was washed once in 20 mM Trisbuffer, pH 7-5, and centrifuged again at 13000 g for 30 min (4 °C). The pellet was then re-suspended in 50 ml of 20 mM Tris buffer containing 0-5 mg DNase (pH 7-5) and centrifugedat 13000 g for 30 min at 4 °C. The supernatant was discarded and the pellet was used forplasma membrane preparation by the 2-phase dextran-polyethyleneglycol method describedby Brunette & Till (1971). Isolated plasma membranes were stored at —20 °C in 30 % glycerol.Protein concentration was determined by the method of Lowry, Rosebrough, Farr & Randall,(1951) with bovine serum albumin as standard.

Electron microscopy

The plasma membrane fraction was pelleted by centrifugation at 60 000 g for 15 min. Thepellet was fixed in 5 % (v/v) glutaraldehyde in o-i M sodium cacodylate buffer, pH73 , for1 h at room temperature, post-fixed with 1 % osmium tetroxide also in cacodylate buffer for1 h, stained with *•$% uranyl acetate, dehydrated through a graded series of ethanol, andfinally embedded in Spurr resin (Spurr, 1968). Thin sections were cut on a LKB ultramicro-tome, and stained with Sato's lead (Sato, 1968). The sections were examined in a Philips 201electron microscope.

Enzyme assays

The marker enzymes alkaline phosphatase (EC 3.1.3.1), succinate dehydrogenase (EC1.3.99.1) and glucose 6-phosphatase (EC 3.1.3.9) were used to assess the purity of the plasmamembrane fraction, as described by Green & Newell (1974).

Alkaline phosphatase was used as a plasma-membrane marker. The rate at which £-nitro-phenol was liberated at pH 10-3 and 22 °C was measured as AA/min at 404 nm. The specificactivity was expressed as /imol liberated/min/mg protein. The molar extinction coefficient for/>-nitrophenol at 404 nm was 1-73 x io4 litre/mol/cm (Bessey & Love, 1952).

Mitochondria were located by assaying succinate dehydrogenase, an enzyme generally con-sidered to be located in the inner mitochondrial membrane (Schneider, 1946). The rate ofreduction of 2,6-dichlorophenolindophenol at pH 72 and 22 °C was measured as A^/minat 600 nm and the specific activity reported as fimol product/min/mg protein. The molarextinction coefficient used for 2,6-dichlorophenolindophenol was 21000 litre/mol/cm (Green& Newell, 1974).

Endoplasmic reticulum was located by determining glucose-6-phosphatase, an enzymeconsidered to be a microsomal marker (Goldfischer, Essner & Novikoff, 1964). Inorganicphosphorus was determined by the method of Fiske & Subbarow (1925). The specific activityof the enzyme was reported as nmol of inorganic phosphorous released/min/mg protein.

Materials

All reagents used were of analytical grade. Digitonin, cycloheximide, dexrran, polyethylglycoland substrates for the enzyme assays were obtained from Sigma (London) Chemical Co. Ltd.

248 A. R. Jaffi and D. R. Garrod

20 40 60 80 100Membrane added, ^g protein/ml

250

Fig. 1. Dose-response curves showing the effect on cohesion of log-phase Ax-2 cells ofplasma membranes isolated from log-phase cells ( • ) , aggregation-competent cells(A), stationary phase cells ( • ) , and. cycloheximide-treated cells (O). Each pointrepresents the average of 8 determinations from 2 experiments using separate batchesof membranes.

RESULTSEffect of isolated plasma membranes on cohesion of log-phase cells

Plasma membranes isolated from log-phase Ax-2 cells gave complete inhibition oflog-phase cell cohesion when added to the cell suspension at 25 /ig membraneprotein/ml (Fig. 1).

According to the suggestion of JafK et al. (1979), stationary phase membranes shouldpossess an incomplete ligand-receptor system and should therefore inhibit log-phasecell cohesion less effectively than plasma membranes from log-phase cells. It wasfound that only partial inhibition of log cell cohesion was achieved even at 250 /tg/mlof stationary cell membrane protein (Fig. 1).

The vegetative cell cohesion mechanism persists on the surface of aggregation-competent cells (Beug et al. 1970). It would therefore be expected that membranesfrom aggregation-competent cells should inhibit log-phase cell cohesion. This was

Plasma membranes and cell cohesion 249

100-1

3 4 5 6 7 8Time of shaking, h

Fig. 2. Effect of cycloheximide on cohesiveness of log-phase Ax-2 cells aged in phos-phate buffer. Cells were shaken in the presence (#) or absence (O) of cycloheximide(500 /ig/ml) and at the times indicated they were harvested, washed in cold distilledwater and their cohesiveness assayed, in phosphate buffer as described in Materialsand methods. The equilibrium particle count after 20 min is plotted on the ordinate.Each point represents the average from 3 experiments.

found to be the case, but complete inhibition was not achieved until the concentrationof aggregation-competent cell membrane protein exceeded ioo/tg/ml (Fig. 1).

When log-phase cells were shaken in suspension for 8 h in the presence of 500 /Jg/mlcycloheximide, complete loss of cohesiveness was observed (Fig. 2). These cells werefound to recover their cohesiveness when incubated overnight in the absence ofcycloheximide. It was suggested by Hoffman & McMahon (1978) that cycloheximide-treated cells (CH-cells) have no CS A or CS B on their surfaces. Thus, plasma mem-branes from CH-cells should be without effect on log cell cohesion. No significantinhibition of cohesion was found even in the presence of 250/ig/ml of CH-cellmembrane protein (Fig. 1).

250 A. R. JfaffS and D. R. Garrod

20 40 60 80Membrane added, pg protein/ml

100 250

Fig. 3. Dose-response curve showing the effect on cohesion of aggregation-competentcells by plasma membrane isolated from log-phase cells. Each point represents theaverage of 8 determinations from 2 experiments using separate batches of mem-branes.

Effect of isolated log-phase cell plasma membranes on cohesion of aggregation-competentcells

Fig. 3 shows that the cohesion of aggregation-competent cells was only partiallyinhibited by the concentration of log-phase cell membranes sufficient completely toprevent cohesion of log-phase cells. Maximal inhibition was achieved at a membraneprotein concentration of 50/ig/ml, and never exceeded 46% even at much higherconcentrations.

Effect of isolated log-phase cell plasma membranes on cohesion of different slime mouldspecies

In order to determine the degree of species specificity among different slime mouldsthe effect of log-phase membranes on the cohesion of feeding cells of Dd NC-4, Dm,Dp, and Pv was tested. Fig. 4 shows that the dose-response curves for inhibition ofcell cohesion versus membrane concentration were very similar for the 4 differentspecies, complete inhibition being achieved in each case.

Purity of plasma membranes

An electron micrograph of the plasma membrane fraction isolated from log-phasecells is shown in Fig. 5. It shows a mixture of membrane sheets and vesicles with arather heterogeneous size distribution. There were no structures recognizable asmitochondria or rough endoplasmic reticulum.

As a further check on the purity of the membrane fraction, marker enzyme activi-ties were assayed on the particulate fraction from the original cell homogenate (after

Plasma membranes and cell cohesion 251

100-1

9 0 -

10

20 40 60 80 100Membrane added, pg protein/ml

250

Fig. 4. Dose-response curves showing the effect on cohesion of feeding cells of differentspecies of slime moulds by plasma membranes isolated from Ax-2 log-phase cells.A, Dictyostelium purpureum; # , D. discoideum strain NC-4; O» D. mucuroides; • ,Polysphondylium violaceum. Each point represents the average of 8 determinations from2 experiments using separate batches of membranes.

the first wash in 20 mM Tris buffer), and the pelleted membrane fraction. The resultsare shown in Table 1. The plasma membrane fraction showed 9/45-fold enrichmentwith respect to alkaline phosphatase, a plasma membrane marker. In the membranepellet the mitochondrial and endoplasmic reticulum markers, succinate dehydro-genase and glucose-6-phosphatase, were respectively depleted to 0-00014 a nd 0*032of their specific activities in the homogenate.

DISCUSSION

A prediction from the ligand-receptor model for the cohesive behaviour of log-phase Ax-2 cells (Jaffa et al. 1979), would be that plasma membranes isolated fromthose cells should bind to the surface of intact log-phase cells by complementarymolecular interaction, thus inhibiting cell cohesion. The present results are consistentwith this prediction. Furthermore, plasma membranes isolated from cells which, inour view, have incomplete (stationary), different (aggregation-competent) or absent

252 A. R. JaffS and D. R. Garrod

Fig. 5. Electron micrograph of isolated plasma membrane fraction from log-phase cells.

(CH cells) molecular cohesion mechanisms were either less effective or completelyineffective in terms of inhibition of cohesion of log cells. Our interpretation of theinteraction of different types of membranes with the log-phase cell surface can bevisualized as shown in Fig. 6.

We now have 2 pieces of evidence which suggest that log-phase cells cohere via asingle mechanism. The low molecular weight inhibitory factor isolated from stationarymedium (Swan et al. 1977; Garrod et al. 1978; Jaff£ et al. 1979) and isolated plasmamembranes from log-phase cells produce 100% inhibition of log-phase cell cohesion.It seems likely that this mechanism is identical with CS B (Beug et al. 1973), though

Plasma membranes and cell cohesion 253

Table 1. Enzymic characterization of isolated plasma membranes

Marker enzyme

Alkaline phosphatasejSuccinate dehydrogenasejGlucose-6-pho8phatase§

Specific

CrudeParticulatefraction*

0-0355 9 0

0-043

activity

CellMembrane

fraction

03310-008o-ooi

Relativespecific

activityf

9-450-000140-032

• 13000 g after washing in 20 mM Tris, pH 7-5.f Ratios of sp. act. in the membrane fraction to the sp. act. in the crude fraction.% /imol substrate utilized/min/mg protein.§ nmol substrate utilized/min/mg protein.Each value represents the average obtained from 3 assays.

Log

AggC CH

Fig. 6. Diagram showing the proposed interactions of plasma membranes isolatedfrom log-phase cells, stationary phase cells, CH cells and aggregation-competentcells, with the surface of log-phase Ax-2 cells. -•)— CS B; —̂— CS A.

at present there is no direct evidence for this. It seems certain, however, that thecohesive mechanism does not involve the carbohydrate-binding protein 'discoidin*(Rosen & Barondes, 1978). This is because our inhibitory factor has been found tobe without effect on the discoidin-mediated agglutination of rabbit erythrocytes(Nossiter & Garrod, Unpublished. A sample of pure discoidin was generously pro-vided by Dr G. Gerisch and his colleagues).

The specificity of the inhibitory effect of isolated log-phase cell membranes hasbeen demonstrated by examining their behaviour with aggregation-competent cells.These membranes produced a maximum of 46% inhibition of aggregation-competentcell cohesion, which is almost identical to that obtained with the low-molecular-weight inhibitory factor previously shown to be specific for log-phase cell cohesion(Jaffe" et al. 1979). This result emphasizes the possibility that the inhibitory factor andthe log-phase cell membranes interact with the same molecular cohesive sites.

Our results with CH-cells and the plasma membranes derived from them, support17 CEL 40

254 A. R. Jafff and D. R. Garrod

the contention of Hoffman & McMahon (1978) that these cells lack contact sites. Inrelation to our work, we wish to stress the difference between CH-cells and stationaryphase cells. We have suggested that the latter possess an incomplete ligand-receptorsystem on their surfaces because, though they do not cohere mutually, they are ableto stick to log-phase cells (JafTe et al. 1979). CH-cells on the other hand, do not stickto log-phase or stationary cells (JafT6, unpublished). This is consistent with the com-plete absence of inhibitory activity of CH-cell membranes on log-phase cell cohesion.

It has been shown previously that autoclaved membranes from aggregation-phasecells of D. discoideum strain Ax-3 inhibit chemotactic aggregation and developmentallyregulated enzyme synthesis (Smart & Tuchman, 1976; Tuchman, Smart & Lodish,1976). It is not clear what these authors believe about the role of cell cohesion in theeffects they describe. In their first paper they state that' the membranes appear to actby preventing cells from becoming competent to aggregate, rather than by simplyblocking the formation of intercellular bonds', while in their second paper they suggestthat, 'the formation and maintenance of specific cell-cell contacts may play acrucial role in the control of slime mould development.' We suggest that the inhibitoryeffect of membranes on development is partly due to the inhibition of cell cohesionwhich we have demonstrated. This is emphasized by our observation that the low-molecular-weight cohesion-inhibiting factor which is specifically active against contactsites B also reversibly inhibits development (Swan et al. 1977; Garrod et al. 1978;JafTe et al. 1979).

It has recently emerged that slime mould cells of different species show mutualadhesion (Nicol & Garrod, 1978; Springer & Barondes, 1978; Bozzaro & Gerisch,1978; Sternfeld, 1979). Earlier work (Raper & Thorn, 1941; Shaffer, 1957a, b; Bonner& Adams, 1958) suggesting that cohesion was strongly species-specific should probablynow be interpreted in terms of chemotactic, rather than adhesive, specificity (Garrod,1974). Our present results suggest that CS B-mediated cohesion is not species-specific because cohesion of vegetative amoebae of 4 species was inhibited with equalfacility by isolated plasma membranes from Ax-2 log-phase cells. This result isconsistent with our previous finding that the low-molecular-weight inhibitory factorisolated from stationary phase medium blocks cohesion of these 4 species (Swan et al.1977). The sorting-out which takes place between different species within aggregatesin suspension (Nicol & Garrod, 1978; Bozzaro & Gerisch, 1978; Sternfeld, 1979) maybe accounted for by the species specificity of the CS A system suggested by immuno-logical techniques (Miiller & Gerisch, 1978), though chemotaxis and differentialadhesion may also be involved.

The chemical characterization of the constituents involved in the cohesion mechan-ism of vegetative cells is now proceeding.

We thank Mrs Hilary Nossiter for technical assistance, and Professor J. M. A. Whitehousefor his comments on the manuscript. This work was supported partly by the Science ResearchCouncil and partly by Research Grant no. 1205 from Consejo Nacional de InvestigacionesCientificas y Tecnologicas (CONICIT), Venezuela.

Plasma membranes and cell cohesion 255

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(Received 10 May 1979)