J. Cell Sci.

38, 283-292 (1979) Printed in Great Britain Company of Biologists Limited igyg

283

CELL SURFACE LIPIDS AND ADHESION

IV. THE EFFECTS OF TRYPSIN ON LIPID TURNOVER BY THE PLASMALEMMA A. S. G. CURTIS AND OYA HILL
Department of Cell Biology, University of Glasgow, Glasgow G12 8QQ, Scotland

SUMMARY

Trypsin treatment of intact cells or isolated plasmalemmae from embryonic chick neural retinae leads to an accumulation of lysophospholipids in the plasmalemmae. Trypsin was used at activities commonly used in cell disaggregation techniques. This accumulation appears to result from the decrease in acyltransferase activity in the plasmalemma produced by enzyme treatment. Plasmalemmal CoA ligase activity is not affected by trypsin treatment. Trypsinization has little effect on plasmalemmal phospholipase As activity. These results are discussed in relation to (a) the effects of trypsinization on cell adhesion, and (b) the theory that cells cannot adhere to lecithins because of their fluidity or surface-free-energy values. We propose that the effects of trypsinization on adhesion may in large part be due to the effects of the enzyme on plasmalemmal enzymes of the lipid turnover system rather than to effects on other plasmalemmal proteins. Similarly the inability of cells to adhere to lecithin substrates is simply explained as being due to the lysolecithin that contacting cells release from these substrates. INTRODUCTION

Although trypsin (EC. 3 . 4 . 4 . 4 ) has been used in pure and impure forms for separating tissues into cells from the time of SchiefFerdecker (1886) and Rous & Jones (1916), we are still unclear as to the mechanism or mechanisms by which it acts in these systems. Edwards & Campbell (1971) showed that the pure enzyme acts in detaching BHK cells from glass and plastic. Moscona (1963) had already shown that soya-bean trypsin inhibitor prevents the enzyme acting in cell separation. However it is uncertain as to which cell component or components have to be damaged by this proteolysis to allow cell separation. It has of course been often assumed that the component is plasmalemmal in site and that it is a protein or proteins directly involved in cell adhesion. Many workers (Cook, Heard & Seaman, i960; Hynes, 1974, for example) have shown that trypsinization releases glycopeptides from the cell surface, though Kraemer (1967) failed to find appreciable release of sialic acid-rich components from the surfaces of CHO cells when they were trypsinized. Rees, Lloyd & Thorn (1977) suggest that trypsin acts both on the general stiffness and shape of a cell as well as on the systems that directly effect the adhesion of part of the plasmalemma to a substrate but did not identify the plasmalemmal component that is damaged by trypsinization. We propose a new hypothesis about one of the actions of trypsin on cell adhesion. This is based in part on the results reported in the 3 previous papers in this series

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(Curtis, Campbell & Shaw, 1975 a; Curtis, Shaw & Spires, 19756; and Curtis, Chandler & Picton, 1975 c). We propose that trypsin affects the status of phospholipids in the plasmalemma and that in turn adhesion depends at least in part on phospholipid status. The term phospholipid status means the amount and type of diacyl phospholipid relative to monoacyl phospholipid (lysophospholipid) and other plasmalemmal components. The results reported in our earlier papers suggested that fatty acid chain length and unsaturation could have substantial effects on adhesion. In the first paper of this series we described the marked similarity in adhesive behaviour of those cells that had been exposed to phospholipase A2 and those that had been treated with trypsin, after previous separation with the chelating agent EDTA in both cases. Cells exposed to EDTA alone adhere in the cold (Curtis & Greaves, 1965) but subsequent trypstnization or exposure to phospholipase or to lysolecithin renders these cells non-adhesive in the cold. This parallel extends to the effects of various metabolic inhibitors on the adhesion of these cells after exposure to trypsin or to phospholipase. At first sight a parallel between trypsinization and lysis of phospholipids must seem improbable. However, in the second paper of this series we showed that the components of a diacyl-monoacyl phospholipid turnover system were present in the plasmalemmae of embryonic chick neural retinal cells. Resch and his co-workers have of course demonstrated a similar system in lymphocytes (Resch, 1976). Thus if either the ligase or the acyltransferase were more sensitive to trypsinization than the phospholipase, trypsin treatment of the cells would lead to the accumulation of lysophospholipids in the plasmalemmae. This accumulation might, following the results reported in our first paper, be expected to reduce cell adhesion. This paper reports measurements made to determine whether phospholipid turnover and the enzymes involved in it are affected by trypsinization of cells or isolated plasmalemmae. The main questions we consider in this paper are as follows. (1) Does trypsinization of cells lead to accumulation of lysophospholipids in their plasmalemmae ? (2) Does trypsinization of cells diminish the overall reacylation of lysophospholipids in the plasmalemma ? (3) Does trypsinization reduce the plasmalemmal coenzyme-A ligase activity ? (4) Does trypsinization reduce the acyltransferase activity of plasmalemmae ?

MATERIALS AND METHODS Neural retina cells were obtained from 7-day-incubated chick embryos (Consort strain) by dissection. The retinae were dispersed into single-cell suspensions with EDTA using the technique described by Curtis et al. 1975 a. Plasmalemmal isolation was carried out using the technique described by Curtis et al. 1975 b, or for those experiments in which lysophospholipid measurement was made using the technique described by Ferber, Resch, Wallach & Imm (1972) with reduction in the times allowed for centrifugation to approximately two thirds of those recommended. Sigma Grade III trypsin was used for the treatment of cells or plasmalemmae, activities were assayed by following the hydrolysis of BAEE. In general phospholipids were separated and identified using the TLC methods described in detail by Curtis et ah 1975 b. Methods B and E described in that paper were chiefly used. Radio-chemicals were obtained from the Radiochemical Centre, Amersham, U.K. or N.E.N. Gmbh.

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Incorporations of radioactivity into TLC spots or into precipitated acyl-coenzyme A compounds were measured using a Beckman CPM zoo liquid scintillation counter with 0-4 % PPO in toluene as the scintillant. The accumulation of lysophospholipids in the plasmalemmae of trypsinized cells was measured by prelabelling known numbers of cells with [14C]stearate (sp. act. 2-5 /tCi /tmol""1) using the incorporation system described in the first paper in the series. The cells were then washed 3 to 5 times with a modified Hanks'-HEPES saline, containing 001 M HEPES at pH 7-4 as a buffer, with the addition of ATP, 1-25 x io~E M dm~3 and CoA, free acid form 5 x io" 8 M dm"1. This saline was used so that free fatty acid formed by the cells from hydrolysis of diacylphospholipids might be reincorporated thus reducing any lysophospholipid accumulation simply due to lack of ATP and CoA required for reacylation. The washing was continued until the level of radioactive stearate in the washing medium fell below 01 % of that in the original incorporation medium. Their plasmalemmae were prepared by the Resch method, they were then trypsinized in the final extraction medium for 20 min at 37 C using 150 BAEE units of trypsin per ml. Phospholipids were extracted from the plasmalemmal fraction and run on TLC silica gel paper using eluants B or E. The position of radioactive spots was determined either by a Panax TLC radioactive scanner or more permanently by preparing autoradiographs using X-ray film in contact with the TLCs. Finally spots were cut out of the TLC sheets and their radioactivity counted by scintillation counting. The effects of trypsinization on reacylation were measured by trypsinizing plasmalemmal fractions or treating whole cells with 400 BAEE units trypsin for 10 min (experimental) or incubating them with o-ooi M EDTA for the same period at 25 CC. Trypsin and EDTA were dissolved in Ca-Mg-free Hanks'-HEPES medium and equal volumes of these media were added to the plasmalemmal aliquots or to the cells. Incubation was terminated by adding 1 mg of soya bean trypsin inhibitor to control and experimental series. An equal volume of incorporation medium was then added to each aliquot containing [14C]oleate (sp. act. 4-0 /tCi /tmol"1) and incorporation was allowed to proceed at 37 C for 20 min. The whole cells were then processed to obtain plasmalemmal fractions. Lipids were then extracted with chloroformmethanol and after concentration of the extracts under dry nitrogen the lipids were run on TLC using methods B, C and E (see Curtis et al. 19756 for details). The effect of trypsinization on the CoA-ligase system was investigated as follows. Plasmalemmal fractions were trypsinized with 400 BAEE units of trypsin at 37 C for 20 min. They were then incubated with [14C]stearic acid (sp. act. 25 /tCi /tmol"1) in the ATP, CoA, Hanks'HEPES incorporation medium for 15 min at 37 C. Stearoyl-CoA was precipitated by acidifying with perchloric acid to 2 % after addition of 2 mg cold stearoyl-CoA and 5 mg bovine serum albumin. The precipitate was pelleted, redissolved in w-butanol, H2O, acetic acid ( 5 : 3 : 2 v/v) and run on Whatman No. 1 chromatographic paper with this solvent in the ascending mode with appropriate standards. The effect of trypsinization on the acyl transferase system was studied by following the incorporation of stearoyl-CoA (14C-labelled, sp. act. 4-9/tCi/tmol"1) into phospholipids by plasmalemmal fractions. The effects of trypsinization on plasmalemmal phospholipases were examined by following the lysis of ([l4C]lecithin (dioleolyl)) layers on a glass substrate forming the walls of the culture dish. Cell contact is necessary to obtain extensive lysis which suggests that the phospholipases involved have to be in contact with the substrate and are on the plasmalemma. Cells or plasmalemmal fractions treated with EDTA in place of trypsin formed the controls to the 4 experimental systems described above.

RESULTS Accumulation of lysophospholipids in the plasmalemma after trypsinization

In order to avoid exchange reactions between the plasmalemmae and internal organelles or the possibility that lysophospholipids might be derived from organelles other than the plasmalemma, the trypsinization was carried out on isolated plas19 CEL 38

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A. S.G. Curtis and O. Hill

malemmae. The plasmalemmae had been previously labelled with [14C]stearate in the CoA-ATP incubation medium while they were still on intact cells. Lysophospholipids and other lipids were extracted and identified by i?i values on TLC. Results are shown in Table i, as dpm after subtraction of background. Clearly it is likely that there has been appreciable loss of lysophospholipids from the plasmalemmae during the incubation but these would be recovered during the extraction of the whole plasmalemmal fraction. It is of course possible that accumulation of lysophospholipids and degradation of plasmalemmal phospholipids may have taken place during
Table i. Levels of phospholipid and lysophospholipids in plasmalemmae of chick neural retinal cells after exposure to trypsin or EDTA medium
Trypsinized A Free fatty acid Lysophosphatidyl serine and ethanolamines Lysophosphatidyl choline Phosphatidyl serine and ethanolamine Phosphatidyl choline Phosphatidyl inositol Phosphatidyl ethanolamine and glycerol Sphingomyelin Neutral lipids Ceramide B 25370 960 508 1046

Control
A

C
1113

A 9369

B 1830
274

C
204

1
1

749i 495 574 660 198 NM 178

34
2475 i960

290 844

2406

NM NM NM NM

1685 904 6010

1366 1686 680

NM NM

2390 224

Cells labelled with [14C]stearate (sp. act. after the addition of unlabelled stearic acid 25 /tCi /tmol" 1 ) in incorporation medium, 2 /tCi per 3 x i o 6 cells. Plasmalemmae extracted and in each experiment divided into equal aliquots, one of which was treated with 150 BAEE units trypsin while controls were treated with E D T A 1 x I O ~ 3 M in Hanks'-HEPES for 2omin. Results are expressed in dpm per 1 x 10' cells. N M , not measured.

the isolation of the plasmalemmal fraction but this does not account for the marked increase in lysophospholipids and diminution in phospholipids found in the trypsinized set of measurements as compared with the control that was incubated in EDTA. It is unlikely that the low levels in the control represent an inhibitory effect of EDTA on phospholipase activity because unincubated controls (Curtis et al. 19756) contain low levels of lysophospholipid.
Inhibition of reacylation by trypsinization

The results shown in Table 1 suggest that trypsinization produces an accumulation of lysophospholipids in the plasmalemma and that these are derived from plasmalemmal phospholipids. Does trypsinization have this effect by stimulating the

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287

degradative part of the turnover system or does it damage the reacylation system ? In order to test this question cells were exposed to trypsin (400 BAEE units/ml) for 10 min at 25 C in a CMF-saline, or to the CMF-saline alone. The cells were washed and then their ability to incorporate [14C]oleate into phospholipids was measured. Counts on the lipids extracted from the plasmalemmal fraction are given in Table 2. A parallel experiment on the ability of isolated plasmalemmae treated either with EDTA or trypsin to reacylate gave results summarized in Table 3. Table 2. Effects of trypsinization on reacylation by whole cells: incorporation into plasmalemmal fractions
Trypsinized Control

0-222 Phosphatidyl cthanolamine and glycerol 0566 Phosphatidyl inositol Undetectable 0313 0-037 0078 Phosphatidyl serine 0-075 Phosphatidyl choline 2698 Measured as incorporation of [14C]oleate into plasmalemmal fraction after trypsinization (experimental) and extraction of lipids and their identification and separation on TLC. Results are expressed as nmol per io7 cells. Sp. act. of oleic acid, 4-0 /*Ci /tmol"1; 34 nmol of oleate added in incubation medium per io7 cells. Results are means of 3 repeats, standard deviations less than 15 % of means.

Table 3. Inhibition of reacylation by trypsinization of plasmalemmae

Trypsinized Phosphatidyl Phosphatidyl Phosphatidyl Phosphatidyl ethanolamine and glycerol inositol serine choline
0-157 0-033 0018 0054

Control
O-II 016 0-003 O-O2O

Measured as incorporation of [14C]oleate into plasmalemmal fraction after trypsinization (experimental) and extraction of lipids and their identification and separation on TLC. Results are expressed as nmol per io7 cells. Sp. act. of oleic acid 4-0/tCi /imol"1; 34 nmol of oleate added in incubation medium per io7 cells. Results are means of 3 repeats, standard deviations less than 15 % of means.

It is clear that overall reacylation is markedly diminished in recently trypsinized cells or plasmalemmae. This could be due either to destruction of one or both of the enzymes involved in the reacylation, or to destruction of the phospholipase or to damage to membrane structures required for the activity of the system. It may seem at first sight a little strange to suggest that inhibition of phospholipase activity might be involved, for this enzyme will be responsible for the appearance of lysophospholipids. However reduced phospholipase activity would diminish the size of the lysophospholipid pool so that there would be less available for reacylation. For this reason we examined the effect of trypsinization on each of the enzymes involved.

19-2

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Effects of trypsinization on piasmalemmal CoA-ligase and on acyl transferase Ligase activity was measured by following the incorporation of [14C]stearate into stearoyl-CoA by trypsinized and by EDTA-treated plasmalemmal fractions. Results are shown in Table 4. Clearly trypsinization actually stimulates CoA-ligase activity. Table 4. Effect of trypsinization on acid CoA ligase activity of neural retinal plasmalemmae
Incorporation of [14C]stearic acid into stearoyl CoA Trypsinized I II III IV V
I-2O 0-29

Control
0-23 0-17 0-17 0-25 0-40

0-47
068 068

Results of 5 experiments, I-V. Incorporations expressed as nmol [14C]stearic acid incorporated in plasmalemmae from 1 x 108 cells in 15 min. Plasmalemmae incubated for 2omin with 400 BAEE units of trypsin at 37 C (experimental) or EDTA (control) and then incubated with incorporation medium. 10 nmol of stearate added per io8 cells, sp. act. 2-5 /tCi /tmol"1.

Table 5. Effect of trypsinization on acyl transferase activity of neural retinal plasmalemmae

Incorporation of [14C]stearoyl CoA into phosphatidyl choline and lysophosphatidylcholine Trypsinized
1

Control
A

II
840 1140

III 3175 1 go
1

I
5980 240

11
496O

III
68O5

Phosphatidyl choline Lysophosphatidyl choline

14

1240 270

398

us

Incorporation of [ C]stearoyl CoA (sp. act. 4-9 /tCi fimol* ) into phosphatidyl and lysophosphatidyl choline. Approximately 25 nmol of stearoyl-CoA incubated with each aliquot of plasmalemmal fraction for 20 min after either trypsinization or control treatment with EDTA 1 x io~ 3 M dm~ 3 in Hanks'-HEPES medium pH 7-4. Trypsinization (a) with 400 BAEE units trypsin for 10 min, (6) with 100 BAEE units for 10 min. I and II are repetitions of (a) and their controls; III is after trypsinization (6). Results expressed as dpm per io 7 cells.

Acyl transferase activity was followed by measuring the incorporation of [14C]stearoyl CoA into phospholipids, in trypsinized and EDTA-treated plasmalemmal fractions. Table 5 shows the results. Trypsinization markedly reduces this incorporation when compared with that observed in EDTA-treated plasmalemmae.

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This enzyme activity was measured using whole cells in contact with a [14C]lecithin surface after EDTA or trypsin treatment of the cells. The activities are given in Table 6.
Table 6. Hydrolysis of lecithin substrate by neural retina cells Total lysolecithin released into:
A. ICclLIilClll f

of cells Trypsinized: Shaken Still EDTA: Shaken Still

Supernatant
55H

Cells
560

40504 31466 26802

37S
991
1121

Total cpm per flask 58500 3. Substrate dioleoyl lecithin, sp. act. 60 fid /tmol-1. Flasks incubated i h at 37 CC. Shaking rate 701pm. Lysolecithin recovered by CHC13: methanol extraction and isolated by TLC method 1. Means of 3 repetitions.

It is clear that after trypsinization the lysolecithin released into the supernatant is appreciably greater (ca. 7 times) when the cells can contact the substrate than when the cells cannot (shaken). The activity is greater than that of either still or shaken EDTA-separated cells - this slight inhibition with EDTA-separated cells may represent either slight inhibition by carry over of EDTA or greater accessibility of plasmalemmal phospholipases to the substrate after trypsinization. More important however are 2 conclusions. First, that trypsinization does not inhibit phospholipase activities that are plasmalemmally associated. Secondly, that after EDTA treatment the phospholipases lose their association with the plasmalemma which was revealed in the experiments with trypsin by the need for cell contact. The results can be summarized by stating that trypsinization has little effect on the phospholipase, stimulates the ligase but inhibits the acyl transferase. This behaviour is consistent with the overall inhibition of reacylation detected in the experiment reported above. It is not entirely consistent with the results reporting the accumulation of lysophospholipids summarized in Table 1 but possibly the apparent losses of both lysophospholipid and phospholipid in trypsinized membranes are to be explained by accumulation of acyl-CoA compounds in the membrane. We were not able to develop an analytical method to detect these components.

DISCUSSION

The results show that lysophospholipids accumulate in the plasmalemma of neural retinal cells when they are trypsinized and that this appears to be due to inactivation of the acyl transferase or alternatively some form of 'burying' of this enzyme as a result of the action of trypsin. The stimulation of the CoA-ligase argues

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that trypsinization of the membrane may in fact make this enzyme more accessible to substrates. The reacylation is probably inhibited by low temperatures and certainly by previous trypsinization of the cells. We are now able to account for the finding that trypsinization inhibits cell adhesion at low temperatures, with a new hypothesis. Moscona (1961) found that trypsinized chick embryonic cells would not form aggregates at low temperatures, though Curtis (1963) and Curtis & Greaves (1965) pointed out that EDTA-separated cells would aggregate at low temperatures. Moscona concluded that the adhesion required a temperature-sensitive synthetic step. These and other related papers, reviewed by Curtis (1973), led Moscona and others to conclude that protein synthesis was required for adhesion. The suggestion followed that the protein(s) synthesized were directly involved in cell adhesion and that perhaps the molecules that bound cell to cell were proteins that could be destroyed by trypsinization. We can now re-examine the interpretations that have been made, of the finding that low temperatures inhibit the adhesion of trypsinized cells. It should first be remarked that with a few exceptions cells that have not been exposed to trypsin will aggregate at low temperatures, see Curtis (1973) for review. It is worth considering whether release of proteases from cells, perhaps during disaggregation, may have an effect similar to that of trypsin, but no evidence on this point appears to be in existence, though of course proteases and trypsin have very similar effects on some features of cell growth and cell surface chemistry, see Reich, Rifkin & Shaw (1975). Moscona (1961) found that trypsinized cells would not form aggregates at low temperatures, and Moscona & Moscona (1966) also found that various inhibitors of protein synthesis or of RNA synthesis would also inhibit aggregation at 37 C. These and other papers by other workers led to the general series of conclusions that (a) protein synthesis was required for the establishment of adhesion, (b) the molecule(s) synthesized were directly involved in cell adhesion, and (c) perhaps the molecules synthesized were directly involved in adhesion as ligands. The first conclusion, with the restriction that the statement applies only to trypsinized cells, is quite correct and the second highly probable, but the third conclusion does not necessarily follow from the evidence. We can now see that trypsinization can act in a different way to prevent adhesion. Trypsin damages the reacylation system and causes the accumulation of lysophospholipids in the cell surface. The lysophospholipids diminish adhesion. Thus untrypsinized cells which have been exposed to lysophospholipids (see Curtis et al. (1975 a) become non-adhesive though there is presumably no proteolytic damage to their surfaces. If cells are incubated warm for a period after trypsinization they repair their reacylation system and reacylate the lysophospholipids to diacyl phospholipids so that the cells become adhesive. So it can now be realized that the effects of trypsinization on adhesion at low temperatures can be sufficiently explained simply in terms of the effects on the lipid status of the plasmalemma and that there is no need to propose that a ligand molecule is damaged by trypsinization. Of course the results do not preclude the possibility that there is simultaneous damage of a ligand molecule but there is no need to propose the existence of such a species.

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It is worth remarking that the proposal made by Curtis (1967) that some of the effects of serum on aggregation could be due to the presence of aggregation-inhibiting protein (AIP) in sera and that trypsin might contain traces of this protein have already, see Curtis et al. (1975 a), been shown to be misconceived. However this series of papers has shown that AIP, which is identified as a phospholipase, phospholipase A2 and trypsin all have similar effects on the lipid status of the membrane though of course phospholipase and trypsin act in different ways to cause the lysophospholipid accumulation. Our finding that lysophospholipids can carry label after the cell has been allowed to incorporate labelled fatty acids was described in the second paper in the series, Curtis et al. (19756). It could be argued that in those experiments which have been carried out on isolated plasmalemmae the situation might be very different from that obtaining in the plasmalemmae of whole cells. Conversely studies made on the plasmalemmae of whole cells might be obscured by reactions taking place elsewhere in the cells and exchanging products with the plasmalemmae. We have endeavoured to cover these 2 different possible sources of error by carrying out some of the reactions both on whole cells and on isolated plasmalemmae (see Tables 2, 3) or by carrying out reactions on whole cells in such a way that the reaction should be limited to the plasmalemma (Table 5). Earlier papers in this series examined the reacylating ability of whole cells and of the effects of this and of lysophospholipids on cell adhesion and produced results which are wholly compatible with those reported here for isolated plasmalemmae of intact cells. Reacylation in isolated plasmalemmae is diminished by trypsinization as it is also in intact plasmalemmae. There is a more extensive reacylation of lysophosphatidyl choline in intact than in isolated plasmalemmae which probably indicates that isolation does do some damage to the system. We would like to draw attention to the work on lymphocyte stimulation by lectins and antigens where stimulation is followed by a remarkable increase in plasmalemmal reacylating activity (Resch, 1976). It is of interest that Evans and Haston (in preparation) have found that stimulation of B lymphocytes from mice is followed by an increase in cell adhesiveness, and that this is independent of any change in the interaction modulation factors described by Curtis & De Sousa (1975). Possibly changes in growth control mechanisms and thus in the cell cycle state of cells may be more widely linked to cell surface lipid changes and to the actions of proteases and phospholipases. The results on the hydrolysis of substrate phospholipid by contacting cells are of interest in several respects. First, if prolonged contact of the cells with the substrate is prevented by shaking, then hydrolysis of the substrate is much reduced compared with that obtaining when prolonged contact is permitted. This statement is true for trypsinized cells whereas hydrolysis by EDTA-treated cells is little different whether contact is permitted or not. This suggests that EDTA treatment may release soluble phospholipases from the cell while trypsinized cells retain a membrane-bound system. Second, it is worth noting that the cells are able to approach the substrate closely enough for membrane-bound enzymes to operate. An alternative though somewhat

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improbable explanation is that the cells can actually phagocytose the substrate but this is unlikely for if it took place label should be found inside the cells: it is not. Third, our finding provides a new explanation for the finding by Maroudas (1973) that cells would not adhere to lecithin. He interpreted this observation in terms of the fluid nature of the egg lecithin he used. We suggest that it is more likely that the failure to obtain cell adhesion on this type of substrate was due to release of lysophospholipids from the substrate by the cells and the consequent reduction in adhesion as the lysophospholipids enter the plasmalemma. REFERENCES G. M. W., HEARD, D. H. & SEAMAN, G. V. F. (i960). A sialomucopeptide liberated by trypsin from the human erythrocyte. Nature, Lond. 188, 1011-1012. CURTIS, A. S. G. (1963). Effect of pH and temperature on cell re-aggregation. Nature, Lond.
COOK,
200, 1235-1236.

A. S. G. (1967). The Cell Surface. London: Logos and Academic Press. A. S. G. (1973). Cell adhesion. Prog. Biophys. molec. Biol. 27, 315-386. A. S. G., CAMPBELL, J. & SHAW, F. M. (1975a). Cell surface lipids and adhesion. I. The effects of lysophosphatidyl compounds, phospholipase A2) and aggregation-inhibiting protein. J. Cell Sci. 18, 347~356. CURTIS, A. S. G., SHAW, F. M. & SPIRES, V. M. C. (19756). Cell surface lipids and adhesion. II. The turnover of lipid components of the plasmalemma in relation to cell adhesion. J. Cell Sci. 18, 357-373CURTIS, A. S. G., CHANDLER, C. & PICTON, N. (1975 c). Cell surface lipids and adhesion. III. The effects on cell adhesion of changes in plasmalemmal lipids. J. Cell Sci. 18, 375-384. CURTIS, A. S. G. & DE SOUSA, M. A. B. (1975). Lymphocyte interactions and positioning. I. Adhesive interactions. Cellul. Inunun. 19, 282-297. CURTIS, A. S. G. & GREAVES, M. F. (1965). The inhibition of cell aggregation by a pure serum protein..J. Embryol. exp. Morph. 13, 309-326. EDWARDS, J. G. & CAMPBELL, J. A. (1971). The aggregation of trypsinized BHK21 cells. J. Cell Sci. 8, 53-72. FERBER, E., RESCH, K., WALLACH, D. F. H. & IMM, W. (1972). Isolation and characterization of lymphocyte plasma membranes. Biochim. biophys. Ada 266, 494-504. HYNES, R. O. (1974). Role of surface alterations in cell transformation: the importance of proteases and surface proteins. Cell i, 147-156. KRAEMER, P. M. (1967). Regeneration of sialic acid on the surface of Chinese hamster cells in culture. II. Incorporation of radioactivity from glycosamine-i-14C. J. cell. Physiol. 69,
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N. G. (1973). Chemical and mechanical requirements for fibroblast adhesion. Nature, Lond. 244, 353-355MOSCONA, A. A. (1961a). Rotation-mediated histogenetic aggregation of dissociated cells. Expl Cell Res. 22, 455-475. MOSCONA, A. A. (1963). Inhibition by trypsin inhibitors of dissociation of embryonic tissue by trypsin. Nature, Lond. 199, 379-380. MOSCONA, M. H. & MOSCONA, A. A. (1966). Inhibition of cell aggregation in vitro by puromycin. Expl Cell Res. 41, 703-706. REES, D. A., LLOYD, C. W. & THOM, D. (1977). Control of grip and stick in cell adhesion through lateral relationships of membrane glycoproteins. Nature, Lond. 267, 124-128. REICH, E., RIFKIND, D. B. & SHAW, E. (1975). Proteases and Biological Control, in Cold Spring Harbor Conferences on Cell Proliferation. Cold Spring Harbor: Cold Spring Harbor Laboratory. 1021 pp. RESCH, K. (1976). Membrane associated events in lymphocyte activation. Receptors and Recognition, Series A, 1, 59-117. Rous, P. & JONES, F. S. (1916). A method for obtaining suspensions of living cells from the fixed tissues, and for the plating out of individual cells. J. exp. Med. 23, 549-555. SCHIEFFERDECKER, P. (1886) Methode zur Isolierung von Epithelzellen. Z. Mikrosk. 3, 483.
MAROUDAS,

(Received 20 November 1978- Revised 17 January 1979)