454

Preliminary

notes

venient or most appropriate. Although Cell-lipid interactions marking cells with different kinds of beads Cell attachment to lipid substrates can be used to identify heterokaryons, it has been our experience that the BUdR/ L. B. MARGOLIS,’ E. V. DYATLOVITSKAYAZand L. D. BERGELSON*, IA. N. Belozersky Laboratory Hoechst technique is easier to use. Further- of Bioorganic Chemistry and Molecular Biology of more, in experiments in which there is a sig- Moscow State University, Moscow, and $M. M. Shemyakin Institute of Bioorganic Chemistry of the USSR nificant amount of cell death (for example, Academy of Sciences, Moscow, USSR fusion with polyethylene glycol [15]>, the use of beads may be unacceptable due to Summary. The features of substrate necessary for cell attachment were studied using different lipid films adcross-contamination of the two populations sorbed on glass coverslips. Mouse embryo ftbroblasts by beads released from dying cells. In such attach and spread on dipalmitoyllecithin, tripalmitin, and sphingomyelin films which were in crystalline (gel) cases, the BUdRlHoechst technique pro- state at 37°C. The liquid-crystalline films made of total vides a useful alternative to autoradio- brain linids. ohosnhatidvlethanolamine. as well as of egg yolk andiat liver lecithins, were non-adhesive for grwhy. cells. Cholesterol which is known to abolish the gel to References 1. Moser, F G, Dorman, B P & Ruddle, F H, J cell bio166 (1975) 676. 2. Latt, S A, Proc natl acad sci US 70 (1973) 3395. 3. -Ibid 71 (1974) 3162. 4. Perry, P&Wolff, S, Nature 251 (1974) 156. 5. Tice, R, Chaillet, J & Schneider, E L, Nature 256 (1975) 642. 6. Ueda, N, Uenaka, H, Akematsu, J & Sugiyama, T. Nature 262 (1976) 581. 7. Y&e, D, Procnatl &ad sci US 61 (1968) 477. Harris, H &Watkins, J F, Nature 205 (1965) 640. f : Moser, G, Muller, H J & Robbins, E, Exp cell res 91 (1975) 73. 10. Hilwig, I & Gropp, A, Exp cell res 81 (1973) 474. 11. Stetten. G. Latt. S & Davidson. R L. Somatic cell genetics 2 i1976) 285 12. Jakob, H, Buckingham, M E, Cohen, A, DuPont, L, Fiszman, M & Jacob, F, Exp cell res. In press. 13. Wright, W E, Exp cell res. In press. 14. Hosli, P, Current trends in sphingolipidosis and allied disorders (ed B W Volk & L Schneck) p. 1. Plenum, New York (1976). 15. Pontecorvo, G, Somatic cell genetics 1 (1975) 397. Received May 13, 1977 Revised version received September 12, 1977 Accepted October 14, 1977

Exp Cell Res 11 I (1978)

liquid-crystalline transition of dipalmitoylle&hin makes it also non-adhesive for the cells. The mechanism of lipid fluid film non-adhesiveness for cell attachment is discussed in relation to cell-cell contact interactions.

The requirement of a solid substrate for cell growth in tissue culture is a significant characteristic of normal cells which is often lost after malignant transformation [I]. Cell attachment and spreading on these substrates are complex events which include outgrowth of cell processes, rearrangement of the cortical layer, assembly of microtubules and formation of microfilament bundles [2-6]. The whole chain of these events is initiated by interaction with the substrate. However, it is not known which particular features of the substrate are required for such initiation and for a successful cell spreading. One way to investigate these features further is to compare adhesive and non-adhesive substrates. Cells are known to attach and spread on substrates of materials such as glass, plastics, metals and collagen. Yet we have found earlier that films made of total bovine brain lipids are non-adhesive for cells [7]. In an attempt to find out which particular properties of the lipid film are responsible for its non-adhesiveness we have investi-

Preliminary

gated cell attachment to films prepared from different lipids. Materials

and Methods

Cells. Mouse (AISnell) embrvo tibroblasts (MEF) of the fist to second passage were prepared‘and cultivated at 37°C on coverslins as described earlier 12.81. The cell media were: Eagle’s media (45 %), lactalbumin hydrolysate (45 %) and bovine serum (10%). The cell cultures were fixed with a Bouin fixator and stained with Mayer hematoxylin. Lipids. The sources of the lipids were as follows: Triolein (Serva Feinbiochemica), egg phosphatidylcholine (lecithin) purified according to [9]; phosphatidylethanolamine, phosphatidylinositol, phosphatidylcholine and phosphatidylserine were isolated from rat liver, as earlier described [lo]. Gangliosides were purified from bovine brain extracts [II]. Two synthetic lipids were used: dipalmitoyllecithin [ 121and sphingomyelin containing stearic acid [ 131. Substrates. Lipid films were prepared by evaporating drops of lipid solution in benzene or chloroformmethanol (20-40 mg/ml) on glass coverslips. After evaporation a thick (Xl-100 urn) lipid film was formed. To increase the stability of the films which at 37°C are below the phase transition point they were incubated for 20 min at a higher temperature than that at phase transition. Assessment of cell attachment. Cells were incubated at 37°C in flasks containing coverslips each with an “island’ of lipid film (about 5-15 mm*). The initial density was approx. 7X lo4 cells/cm2. Most cells attached to the adhesive substrates during the first hour after seeding. However, we counted the number of attached cells 16 h later, in order to promote cell attachment to weakly adhesive substrates possible. At the end of the cultivation eriod the cells were washed in Hanks’ solution and ?txed. In some experiments special flasks instead of common flasks were used for phase contrast observation [8]. It turned out that the fixation procedure did not alter the number of cells attached to the lipids. A lipid film was regarded as non-adhesive when the cell densitv on the film did not exceed 5X lo-*% of that on the glass surface surrounding the film, i.e. when onlv a few cells could be found on the lipid film. The film was regarded as adhesive if the cell density was not lower than 20%, as compared with that on the glass. All films examined in our experiments proved to belong either to adhesive or to non-adhesive substrates, as defined above. Among our materials there were no films with “intermediate” adhesivity: i.e., when the cell density was lower than 20 %, bui higher than 5x 10-Z%. Obviously assessment of the cell adhesion in terms of density on the lipid surface is, at best, semiquantitative. Nevertheless, it was sufficient to reveal the differences between two classes of surfaces observed in our experiments, classified as “adhesive” and “non-adhesive”.

notes

455

Results and Discussion

The results of testing the cell attachment to different lipid films are presented in table 1. Lecithins from two different sources (egg yolk and rat liver), as well as the films of total brain lipids, proved to be nonadhesive. This was also true for phosphatidjlethanolamine and triolein. On the contrary , the films of dipalmitoyllecithin, sphingomyelin and tripalmitin were adhesive for the fibroblasts. During 16 h after seeding the cells could not perform more than one mitotic cycle. Since the cell number on adhesive and nonadhesive substrates differed approximately by a factor of IV, this difference could not be explained by selective proliferation of cells on adhesive substrates. In all experiments cells on the control parts of the glass near the lipid film were attached, spread, and were found morphologically normal. Cell spreading varied on adhesive films formed by different lipids. Table 1. Adhesivity of lipidfilms embryo fibroblasts a

for mouse

Lipid

Adhesivity’ State at 37°C

Total brain lipids Triolein Egg lecithin Rat liver lecithin Phosphatidylethanolamine Dipalmitoyllecithin +cholesterol (1 : 1 mol/mol) Dipalmitoyllecithin Tripalmitin N-Stearoylsphingomyelin

-

Liquid-crystalline Liquid Liquid-crystalline Liquid-crystalline

-

Liquid-crystalline

+ +

Liquid-crystalline Crystalline (gel) Crystalline (gel)

+

Crystalline (gel)

a Drops of lipids in organic solution were evaporated on glass coverslips. The cells were seeded in flasks containing the films and fixed 16 h later. The film was regarded as non-adhesive if the cell density was less than 5X 10e2%of that on control glass. The cell density on the adhesive substrates listed was not less than 20 % of that on control glass. b For a review of the physical state of lipids see [27]. Exp Cell Res III

(1978)

456

Preliminary notes

Cells on a dipalmitoyllecithin layer were not well spread and were not polarized. They were attached by cylindrical processes (3-5 pm) and lamellar cytoplasm and resembled cells on glass in the first stages of spreading [2, 51. A considerable number of cells were spread and polarized on sphingomyelin. On tripalmitin the majority of cells were spread and did not differ morphologically from those on the control parts of the glass. The cells spread on the lipids mentioned above were actually anchored to the lipid films, and not to the underlying glass: when films with spread cells were detached from the coverslip and allowed to float in the media they remained spread and did not contract and round up for at least l-2 h. We also tried to investigate the adhesiveness of several other lipids not mentioned in table 1, i.e. cerebrosides, gangliosides, phosphatidylserine and phosphatidylinositol. But the films made from these lipids were not stable in aqueous solution. Different factors were regarded by different authors as critical for substrate adhesion: the charge of the substrate surface [14, 151, its hydrophilic properties [16, 171, the surface microrelief [18, 191 and the physical state of the substrate [20]. We studied the role of some of these factors using lipid films as substrates. Data presented in table 1 show no correlation between the polarity ,of the lipids, which determines the hydrophilic properties of the film, and their adhesiveness. The cells did not attach to non-polar triolein, but attached and even spread on films of equally non-polar tripalmitin. The films of polar sphingomyelin or dipalmitoyllecithin were adhesive, while those of equally polar egg lecithin or phosphatidylethanolamine were non-adhesive. Unfortunately, our experiments did not Permit any conclusion about the role of the Exp Cell Res I I I (I 978)

charge of the lipid films, since negatively charged lipids did not form stable films. Different lipid membranes have a different microrelief. It seems unlikely, however, that this causes differences in cell adhesion, because cell attachment to “smooth” surfaces is generally higher than to “rough” ones [18]. Films of egg and rat liver lecithins, as well as phosphatidylethanolamine and triolein films, seem smoother than dipalmitoyllecithin, tripalmitin and sphingomyelin films and could thus be expected to be more adhesive, but in fact the opposite proved true. The data in table 1 permit the conclusion that in the case of lipid films the fluidity of the lipids is a property critically determining cell attachment. Cells do not adhere to lipid films which are above the phase transition at 37°C and do adhere to films of sphingomyelin, tripalmitin and dipalmitoyllecithin which are in a crystalline state at 37°C. It is known that addition of cholesterol to phospholipid membranes alters the membrane fluidity. In particular, an equimolar amount of cholesterol abolishes the gel to liquid-crystalline transition of dipalmitoyllecithin, producing a condition of “intermediate fluidity” [21]. In our experiments films prepared from a mixture of dipalmitoyllecithin and cholesterol (1: 1 mol/mol) proved to be nonadhesive for mouse fibroblasts in contrast to films of pure dipalmitoyllecithin having a phase transition at 41°C. It thus appears that cell adhesion depends critically on the physical state of lipids (cf [20]). The fluidity of lipid films could affect cell attachment both indirectly (for example due to different adsorption of “cell spreading factor” on fluid and solid lipids [22, 231) as well as directly through interaction with cell outgrowths.

Preliminary notes As far as intracellular tension is generated in cells after spreading (probably by microfilaments [4, 24]), these tension forces must be counteracted by forces of attachment to the substrate of various parts of the cell surface. We assume that the cellular processes touching the surface of a liquid-crystalline lipid film do not form stable contacts with the components of the substrate due to the high lateral mobility of the latter. The processes which are not anchored on the substrate are withdrawn. The cell can thus neither attach to such substrate nor spread on it. When the lipids of the film are in a crystalline state the cell processes attach firmly to the film surface and do not withdraw. The liquid-crystalline state of the lipid parts of the plasma membranes is at present firmly established and should be taken into consideration when interpreting the possible mechanism of cell-cell adhesiveness. According to the present results the lipid regions of plasma membranes should be non-adhesive for other cells. Thus, some special local alterations of the membrane preventing the lateral movement of its components may be required for the formation of cell-cell contacts, e.g. “anchoring” of membrane components to cortical structures [25] and/or changing the lipid fluidity

D61. By altering lipid films one can mimic different features of plasma membranes which are significant in cell-cell contact interactions . The authors are grateful to Professor Yu. M. Vasiliev and Professor I. M. Gelfand for fruitful discussions and helpful criticism.

457

Olshevskaya, L V, Rovensky, J A, Vasiliev, Y M & Gelfand, I M, Proc natl acad sci US 69 (1972) 248. 3. Rajaraman, R, Rounds, D E, Yen, S P & Rembaum, A, Exo cell res 88 (1974) 327. 4. Goldman, R D, Schloss, J A & Starger, J M, Cell motility. Cold Spring Harbor symp 3 (1976) 217. 5. Bragina, E E, Vasiliev, Y M & Gelfand, I M, Exp cell res 97 (1976) 244. 6. Lazarides, E, Cell motility. Cold Spring Harbor symp 3 (1976) 347. 7. Ivanova, 0 Y & Margolis, L B, Nature 242 (1972) 200. 8. Vasiliev, Y M, Gelfand, I M, Domnina, L V, Ivanova, 0 Y, Komm, S G & Olshevskaya, L V, J embryo1 exp morph01 24 (1970) 625. 9. Dawson. R M C, Biochemj 88 (1%3) 414. 10. Dyatlovitskaya, E V, Yancheskaya,‘G V, Kolesova, N P & Bergelson, L D, Biochimia 38 (1973) 749. In RussianL, Methods carbohydr them 6 11. Tlv;2y;glm, 12. Gordon, D T & Jensen, R G, Lipids 7 (1972) 261. 13. Zvonkova, E N, Mizner, B I, Bushnev, A S, Eller, K I, Soldatova, S A & Evstigneeva, R P, Khimiya orirod soed (1974) 553. In Russian. 14. Macieira-Co&ho,’ A & Avrameas, S, Proc natl acad sci US 69 (1972) 2469. 15. Weiss, L & Harlos, S P, J theoret biol 37 (1972) 169. 16. Baier, R E, Shafrin, E A & Zisman, W A, Science 162 (1968) 1360. 17. Grinnell, F, Miriam, M & Srere, F A, Biochem med 7 (1973) 87. 18. Bershadsky, A D & Lustig, T M, Tsitologiya 17 (1974) 639. In Russian. 19. Trinkaus, J P, Major problems in developmental biology (ed M Locke), p. 125. Academic Press, New York (1966). 20. Maroudas, N G, Nature 244 (1973) 353. 21. Ladbrook, B D, Williams, R M & Chapman, D, Biochim biophys acta 150(1968) 333. 22. Rosenberg, M D, Biophys j 1 (1960) 137. 23. Grinnell, F, Exp cell res 102(1976) 51. 24. Harris, A K, Locomotion of tissue cells (ed M Abercrombie) vol. 14, p. 3. Ciba Foundation symp, Amsterdam, London, New York (1973). 25. Vasiliev, Y M, Gelfand, I M, Domnina, L V, Lubimov, A S & Zacharova, 0 S, Proc natl acad sci US 73 (1976) 4085. 26. Inbar, M, FEBS lett 67 (1976) 180. 27. Phillips, M C, Progress in surface and membrane science 5 (1972) 139. Received May 25, 1977 Revised version received October 25, 1977 Accepted October 3 1, 1977

References 1. Freedman, V & Shin, S, Cell 3 (1974) 355. 2. Domnina, L V, Ivanova, 0 Y, Margolis, L B,

Exp CdRcs

111 (1978)