Lipid composition of plasma membranes from developing chick muscle cells in culture

312

Biochimica et Biophysics Acta, 360 (1974) @ Elsevier Scientific

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LIPID COMPOSITION OF PLASMA MEMBRANES CHICK MUSCLE CELLS IN CULTURE

CLAUDIA

KENT,

STEVEN

D. SCHIMMEL

and P. ROY

FROM

DEVELOPING

VAGELOS

Department of Biological Chemistry, Division of Biology and Biomedical Sciences, Washington University, St. Louis, MO. 63110 (U.S.A.) (Received

March

28th,

1974)

Summary The lipid composition of the plasma membrane and crude particulate fractions from cultured chick muscle cells has been determined. The plasma membrane and crude particulate fractions were isolated from cells grown in high calcium medium to permit nonsynchronous fusion, from cells made to fuse synchronously by growth in low-calcium medium which was then changed to high-calcium medium to initiate synchronous fusion, and from cultures enriched for myotubes by growth in the presence of 5-fluorodeoxyuridine. The lipid to protein ratio in the plasma membranes was unusually high (2.3-2.8, w/w). The major lipid constituents of the plasma membrane were phospholipids (1.8 pmoles/mg protein), cholesterol (0.6 pmole/pmole phospholipid), and tri- and diglycerides (0.03-0.08 ~mole/~mole phospholipid). The phospholipids were composed of approx. 50% phosphatidylcholine, 30% phosphatidylethanolamine, 11% sphingomyelin, 6% phosphatidylinositol, and 3% phosphatidylserine. The fatty acid compositions of the plasma membranes were similar to those of the crude particulate fractions except that the plasma membrane fatty acids were slightly more saturated. No gross differences were observed in the lipid compositions of plasma membrane or crude particulate fractions from myoblasts, fusing cells, and myotubes.

Introduction Embryonic chick muscle cells can be grown in cell culture and provide an excellent system for the study of cellular development and cell fusion. The muscle cells proliferate in culture, then cease division and fuse to form multinucleated myotubes which synthesize muscle-specific proteins and eventually contract spontaneously [ 11. The muscle cells can be made to fuse synchronously by altering the calcium levels in the culture medium [2] and the cells can therefore be isolated and characterized at several stages of development. It is reasonable to expect that differentiation necessitates alterations in the compo-

313

sition of the plasma membrane as the functions of the cell surface change during development. The processes of muscle cell recognition and fusion undoubtedly involve changes in the plasma membrane; the fusion process itself must require at least a local alteration in the structure of the two fusing membranes [3]. We have, therefore, undertaken a study of the plasma membranes of differentiating muscle cells. We previously reported a procedure for the isolation of purified muscle cell plasma membranes based on the identification in the purified fraction of the surface membrane markers, Na’,K’-ATPase and a-bungarotoxin receptors [4]. The present paper describes the lipid composition of the plasma membranes isolated from myoblasts, fusing cells, and myotubes. No gross alterations were observed in the total content of phospholipid, cholesterol or neutral glycerides, or in the total fatty acid or phospholipid compositions. Methods Culture

conditions

Procedures and media for the culture of embryonic chick muscle cells have been described previously [4,5] . Low-calcium medium contained 82% Eagle’s minimum essential medium, calcium and magnesium free; 8.3% horse serum (Grand Island Biological Co., Grand Island, New York); 8.3% embryo extract; 680 ,uM MgS04 ; 200 FM ethyleneglycolbis@-aminoethyl ether) N,iVtetraacetic acid (EGTA); 1% antibioticantimycotic solution (Grand Island Biological Company). The calcium contents of horse serum and embryo extract were determined by atomic absorption spectroscopy on a Perkin-Elmer model 303 spectroscope according to manufacturer’s instructions. The final calcium concentration in the low-calcium medium was calculated to be 160 PM. To restore high-calcium to the culture medium, 1.8 pmoles of CaClz were added per ml of low-calcium medium. The final calcium concentration in this medium was 1960 PM. The medium used for continuous growth in high-calcium contained 1860 E.IMcalcium. Membrane

and lipid isolation

Crude particulate and plasma membrane fractions were isolated as described [4] with the following modifications: the sucrose density gradient consisted of 0.5 ml 55%, 3.5 ml 32%, 4.0 ml 27%, and 2.5 ml 13% (w/w) sucrose, and the density gradient centrifugation was carried out in the SW 41 rotor at 206 000 X g for approx. 16 h. The increase in specific activity of the ouabain-sensitive Na’,K’-ATPase was used to assess purity of the fractions containing plasma membranes. Analysis

of phospholipid

and neutral lipid compositions

Because of the limited quantities of membrane obtainable from cell cultures, it was desirable to facilitate analysis of the phospholipid composition by labeling the phospholipids with a radioactive isotope. The radioactivity of the individual phospholipids could then be determined after thin-layer chromatography. When cells were grown in the presence of 3 ‘Pi, [ ’ 4 C] acetate, [ 3H] glycerol, or [ ’ 4C] glucose during the entire time of culture (about 70 h),

311

the specific radioactivity of the individual phospholipids differed considerably due to variable uptake of lipid precursors from the medium and variable turnover rates of different lipids. Therefore a chemical determination of each phospholipid class was required. The phospholipids were labeled with ’ 2P, in order to locate them by autoradiography. To label the phospholipids with ’ 2P, (New England Nuclear, Boston. Mass.), 15-50 PCi per ml culture medium was added to the cell cultures 20 h prior to harvest. The phospholipids were separated by two-dimensional thinlayer chromatography on silica gel plates containing magnesium acetate (RediCoat, Supelco. Bellefonte, Pa.) using the following solvent systems [6] : chloroform-methanol-28N ammonia (65 : 30 : 5 by vol.) in the first dimension and chloroform-acetone-methanol-acetic acid-H, 0 (3 : 4 : 1 : 1 : 0.5 by vol.) in the second dimension. The phospholipids were located by autoradiography, then elutetl and washed by the procedure of Breckenrldgr et al. 171. Losses of phospholipid were accounted for and corrected by determining the radioactivity remaming in the silica gel after elution and in the upper phase after the wash. Neutral lipids were chromatographed as described by Skipski and Barclay [8] on silica gel H plates using two solvent systems in one direction: first, isopropyl ether-acetic acid (96 : 4 by vol.); second, petroleum ether-diethyl ether-acetic acid (90 : 10 : 1 by vol.). The spots containing the neutral lipids were scraped and the amount of lipid present was determined by the dichromate oxidation procedure [ 91 as described by Skipski and Barclay [ 8 ] . Fatty

acid analysis

Fatty acid compositions were determined by gas-liquid chromatography of fatty acid methyl esters on a 6 foot X 3/16 inch column packed with 10% diethyleneglycol succinate on Anakrom SD 60/70 mesh (Analabs, North Haven, Ct.). Gas-liquid chromatography was performed in a Varian-Aerograph instrument of the 2100 series using the flame-ionization detector. He was used as carrier gas at a flow rate of 30 ml per min. The column temperature was maintained at 150°C until the methyl linoleate peak was eluted. at which time the temperature was raised to 180°C in order to speed elution of the longerchain fatty acid esters and to sharpen these peaks sufficiently to allow accurate measurement. The change in column temperature was shown to have no effect on detector response. The peaks were quantitated by cutting and weighing Xerox reproductions. The fatty acid methyl esters were prepared by acidcatalyzed methanolysis of lyophilized cell fractions. To each dried sample in a screw-cap extraction tube was added 0.1 ml chloroform-methanol (1 : 2 by vol.) followed by 1 ml methanol-HCl (23 : 1 by vol.). The mixture was flushed with N2 and incubated for 16 h in a heating block at 75°C. Water (4 ml) was added and the esterified fatty acids were extracted into three 2 ml portions of diethyl ether. The combined ether extracts were washed once with 2 ml 2% NaHCO 1, the solvent was evaporated under a stream of N2 at O”C, and the esters were dissolved in CS, for injection. This procedure was shown to give essentially quantitative yields of fatty-acid methyl esters from free fatty acids, neutral glycerides, cholesterol esters, phospholipids and sphingolipids.

315

Assays Protein was determined by the method of Lowry, et al. [lo] with bovine serum albumin (Pentex; Miles Laboratories, Inc., Kankakee, Ill.) as standard. Ouabain-sensitive Na’,K’-ATPase was assayed as described previously [4] . Total phosphate was measured by the method of Ames [ll] . The total lipid content of lipid extracts was determined by the method of Marzo et al. [ 121, except that the total reaction volume was reduced to 0.5 ml and the final absorbance was measured at 295 nm to increase the sensitivity of the determination considerably. Standards for this assay were lipid extracts from particulate homogenates whose dry weight had been determined using a microbalance. Total cholesterol was determined spectrophotometrically by the o-phthalaldehyde method [13] using a final reaction volume of 0.6 ml. For the assays of both neutral glycerides and free fatty acid, an appropriate amount of total lipid was applied to a silica gel G thin-layer plate (250 pm) which was then developed in petroleum ether-diethyl ether-acetic acid (90 : 10 : 1 by vol.). The triglyceride, diglyceride and free fatty acid spots were scraped and eluted with 4 ml chloroform-acetic acid (100 : 0.05 v/v). The glyceride eluates were washed once with 0.01 M NaOH-0.1 M NaCl and once with water, then evaporated to dryness, saponified, and assayed enzymatically using the reagents and procedure supplied in the Boehringer test kit for neutral fats and glycerol (cat. no. 15904, Boehringer, Mannheim, Germany). The final reaction volume was 0.3 ml. The free fatty acid eluates were washed twice with water and then assayed by the method of Mahadevan, et al. [14], using a final reaction volume of 0.6 ml. Values of triglyceride and free fatty acid were corrected for losses as determined by the addition of glyceryl tri[9,10-3H] oleate and [l- ’ 4 C] oleic acid to the mixture spotted for thin-layer chromatography. Radioactivity was measured using a 3a70 liquid scintillation cocktail (Research Products Intemational Corp., Elk Grove Village, Ill.) in a Packard Tri-Carb Liquid Scintillation Spectrometer equipped with an Automatic Activity Analyzer. Results Embryonic muscle cells were made to undergo synchronous fusion by manipulating the concentration of calcium in the culture medium. Cultures grown continuously for 66 h in the presence of 1860 PM calcium contained broad, multinucleated myotubes indicating that extensive cell fusion had occurred (Fig. 1). In contrast, no fusion took place in cultures grown for 66 h in low calcium (160 PM) medium (Fig. 2) [ 21. Short, rounded and long spindleshaped cells were prevalent in these cultures. About 70 h after plating, the calcium concentration was increased to 1960 PM to initiate rapid cell fusion. Within 20-30 min some fusion was detectable by electron microscopy (Bischoff, R., personal communication). After l-2 h most of the cells were lined up and long, thin myotubes had formed (Fig. 3). By 3.5-4.5 h (Fig. 4) extensive fusion had occurred, and multiple nuclei were readily apparent in the myotubes, although the myotubes were thinner than those in cultures grown continuously in the presence of 1860 PM calcium. Plasma membranes were isolated from muscle cell cultures grown continuously in the presence of 1860 ,uM calcium to permit non-synchronous

316

Figs 1-4. Phase contrast photomlcrographs (X SO) of embryonic chick muscle cells after three days in culture. (1) Cells grown contmuously in medium containing 1860 FM calcnm~. (2) Cells grown in medium containing 160 IJM calcium. This photograph was taken immediately before addition of high-calcium to the medium. (3) Same culture as in (2). 1.5 h after increase of calcium concentratmn to 1960 PM. (4) Same culture as in (2) and (3). 4.5 h after mcrease of calcium concentration to 1960 @M.

fusion, from unfused cell cultures grown in low-calcium medium (160 PM), from cultures in which synchronous fusion was initiated by increasing the calcium concentration to 1960 PM, and from cultures enriched for myotubes by growth in the presence of 10 ,uM 5-fluorodeoxyuridine [ 151. The lipids were extracted from the crude particulate fraction of the cell homogenates and the purified plasma membranes, and the contents of total lipid, phospholipid, cholesterol (free plus esterified) and neutral glycerides were determined (Table I). The plasma membranes had a very high content of total lipid and phospholipid compared to the crude particulate fractions. In both the plasma membranes and crude particulate fractions the phospholipids accounted for 50-60% of the total lipids. The lipid from the plasma membrane8 contained a higher percentage of cholesterol and less neutral glyceride than the lipid from the particulate homogenates. Free fatty acids accounted for about 3% of the

317 TABLE I LIPID CONTENTS 0~ PLASMA MEMBRANE AND CRUDE PARTICULATE

FRACTIONS

High-calcium and low-calcium cultures were grown for 66 h in medium containing 1860 PM or 160 PM calcium, respectively. High-calcium (1960 pM) was added to low-calcium cultures 1.5 or 4 h before harvest. To some of the high-calcium cultures. 5fluorodeoxyuridine (10 PM) was added 40 h after plating, and the cultures were harvested 88 h after plating. Plasma membrane and crude particulate fractions were isolated and protein and lipid contents were determined as described in Methods. The numbers listed are the averages of two or more determinations. Plasma membrane Na+,K+-ATPase was purified 5.6-10.4-fold with respect to the total homogenate.

Crude particulate fraction High-Ca’+ 5-fluorodeoxyuridine Low-Ca*+ Low-Ca2+* then high-Ca*+ added 1.5 h 4 h Plasma membranes High-Ca* 5-fluorodeoxyuridine Low-Ca2+ Low-Ca*+. then highCaB 1.5 h 4h

mg Total lipid per mg Protein

wmoles Phospholipid per mg Protein

pmoles Cholesterol per pmole Phospholipid

/.tmoles Glyceride* per jlmole Phospholipid

0.48 0.79 0.59

0.41 0.50 0.40

0.41 0.46 0.41

0.12 0.11 0.06

0.67 0.83

0.50 0.51

0.46 0.39

0.12 0.08

2.3 2.8 2.3

1.67 2.01 1.77

0.61 0.55 0.58

0.07 0.04 0.04

2.6 2.6

1.79 1.82

0.57 0.57

0.05 0.08

added

* Triglyceride plus diglyceride

total lipid (data not shown) from both the crude particulate and plasma membrane fractions. There was no detectable monoglyceride. The content of total phospholipid, cholesterol, neutral glyceride, and free fatty acid of either the plasma membrane or crude particulate fractions did not differ significantly with relation to the extent of fusion, although there was a small increase in the total lipid to protein ratio in the particulate fractions as the myoblasts developed into myotubes. The phospholipids, cholesterol, neutral glycerides, and free fatty acids accounted for only 80-85% of the total lipid in both the plasma membrane and crude particulate fractions. To ascertain the nature of the remaining lipids the crude particulate-fraction lipids from 3-day old cells grown in high calcium were chromatographed on thin-layer plates to separate the lipid classes, and the content of each neutral lipid class was determined by dichromate oxidation. By this procedure the lipids that were previously unaccounted for were found to consist of several unidentified neutral lipid components which together composed 14% of the total lipid. The phospholipid compositions of the crude particulate and plasma membrane fractions isolated at the different stages of fusion were determined, and the results are shown in Table II. Phosphatidylcholine plus phosphatidylethanolamine accounted for 75-80% of the phospholipids of both the plasma membrane and crude particulate fractions. The content of phosphatidylserine

318 TABLE

II

PHOSPHOLIPID

COMPOSITIONS

Cultures were grown as described m Table I. Phosphohplds were extracted from the plasma membrane and crude particulate fractions, separated by thm-layer chromatography. and measured by phosphate analysis as described in Methods. The numbers refer to percent of total phospholipid. The phospholipid composltion was determmed on two or more separate preparations for each culture condition, except for 1.5 h and 4 h. The values hsted for the plasma membranes were obtamed from smgle preparations with the most highly purified Na+.K+-ATPase actwity, which was from 7.2-8.9-fold purified with respect to the total homogenate. Phospha-

Phosphatldyl choline

Phosphatidy1 serme

25.8 30.6

58.9 50.7

2.6 3.8

31.3 26.1 30.8

43.9 50.2 51.1

6.9

31.4 32.6

44.2 46.5

tidy1 ethanolamlne Crude particulate High-Ca2+ Low-Ca2+

Sphmgomy&n

Pnosphatldyl mosltol

Cardwhpm

5.1 6.6

5.7 5.9

1.9 2.3

11.2 10.9 11.0

5.0 5.9 4.8

0.2

4.3 2.0

<::;

6.7 2.3

11.4 11.1

6.0 6.7

co.2 <0.2

fractions

Plasma membranes High-Ca*+ 5-fluorodeoxyurldme Low-ca*+ Low-Ca2+, then high-Ca*+ added 1.5h 4 h

_____~~

TABLE

III

FATTY

ACID COMPOSITIONS

._

Cultures were grown as described m Table I. Fatty acid methyl esters were prepared from lyophilized plasma membrane and crude particulate fractions by acid-catalyzed methanolysis and analyzed by gashqud chromatography as described in Methods. The numbers refer to percent of total fatty acids and are the averages of two analyses of a single preparation. Plasma membrane Na+.K+-ATPase was purified 6.4-9.7fold wth respect to the total homogenate. Fatty acid -~--

Crude particulate

16:0

16:l

18:0

18:l

18:2

20:4

Other*

ob Saturated

0.6 0.6

18.8 19.5

1.8 2.3

20.4 20.4

17.5 18.3

18.0 16.5

12.9 13.2

10.2 9.1

39.8 40.5

0.7 0.6 0.5

19.0 19.7 18.2

2.6 1.9 1.9

21.4 21.1 21.0

18.2 18.3 18.0

15.5 16.6 17.4

13.1 12.2 13.4

9.7 9.7 9.8

41.1 41.4 39.7

0.7 0.7

22.9 23.2

1.8 1.6

20.1 19.8

16.7 14.4

14.4 11.6

10.5 12.3

12.8 16.3

43.7 43.7

1.2 0.5 0.6

24.0 23.0 20.5

1.9 1.6 1.6

20.2 21.9 19.0

16.1 14.3 15.2

11.8 12.2 17.0

10.6 11.5 12.1

14.2 15.0 13.9

45.4 45.4 40.1

0.5

11.7

2.4

13.7

19.5

28.7

6.8

10.3

31.9

fraction

High-Ca*+ Low-ca*+ Lsw-ca*+.

then high-Ca*+ added 2h 6h 5-fluorodeoxyuridine

Plasma membranes High-Ca2+ Low-ca*+ Low-Ca*+, then high-Ca*+ added 1.5 h 4 h 5-fluorodeoxyuridine Culture medium * Composed

____~.

14:0

predominantly

of C22 polyunsaturated

fatty acids.

319

was somewhat variable, but this variation showed no reproducible pattern. The percent sphingomyelin in the plasma membrane phospholipids was almost twice that of the crude particulate fraction. None of the phospholipids showed a significant increase or decrease upon fusion of myoblasts to form myotubes. The total fatty acid compositions of plasma membrane and crude particulate fractions were determined by gas-liquid chromatographic analysis (Table III). No significant differences were detected in the fatty acid compositions of the crude particulate fractions. The degree of saturation remained constant at 40-42s in myoblasts, myotubes and in synchronously fusing cells. The fatty acid compositions of the membranes were different from those of the crude particulate fractions. The membranes had a slightly higher level of saturated fatty acids, which was due to increased levels of palmitate. There were more CZ2 polyunsaturated fatty acids and less oleate and linoleate in the membranes relative to the crude particulate fractions, although the total number of double bonds was almost the same. Among the plasma membrane fractions, there was little variation in fatty acid compositions. Membranes from 4-day cultures grown in the presence of 5-fluorodeoxyuridine contained a slightly decreased level of palmitate and a markedly increased level of linoleate compared to the other plasma membranes analyzed. It is likely that these differences are a function of culture age (88 h versus 66 h) rather than extent of fusion. We have found (unpublished results) that the fatty acid compositions of crude particulate fractions of muscle cells grown in low calcium, high calcium, or high calcium plus 5-fluorodeoxyuridine all show a dependence on the age of the cultures and appear to reach constant values after about 4 days in culture. As the cells grow, their fatty acid compositions approach but do not reach the composition of the medium (Table III, bottom line). Discussion The plasma membranes from developing muscle cells in tissue culture have been characterized with respect to content of total lipid, total phospholipid, and several neutral lipids, and the phospholipid and total fatty acid compositions have been determined. The plasma membranes had very high lipid to protein ratios (2.3-2.8 by wt) compared to those reported for plasma membranes isolated from other cells such as liver, HeLa and L cells [ 161. The lipid to protein ratios estimated from data reported for plasma membranes from adult skeletal muscle [17-211 range from 0.23 [17,18] to about 1.5-2.0 [ 191. Plasma membranes isolated from cultured chick embryo fibroblasts had a very high lipid content as indicated by the ratio of phospholipid plus cholesterol to protein of 2.8 (w/w) [22]. It may be that the high lipid content of plasma membranes from cultured chick muscle cells is a property of plasma membranes from avian embryos and not of striated muscle cells in general. The molar ratio of cholesterol to phospholipid was higher in the plasma membranes than in the crude particulate fractions, and this agrees with the findings of others that cholesterol is generally more concentrated in the plasma membrane than in intracellular membranes. The cholesterol to phospholipid ratio in the muscle plasma membranes was similar to values reported for rat liver plasma

320

membranes [16] and rat and rabbit muscle plasma membranes [20,21]. The phospholipid composition of the chick muscle crude particulate fraction was almost identical to that reported for human and mouse skeletal muscle [23]. The plasma membrane phospholipid composition was different from that reported by Fiehn et al. [20], for rat skeletal muscle sarcolemma in that the chick muscle membranes contained more phosphatidylethanolamine and much less phosphatidylserine. The increase in sphingomyelin in the plasma membranes relative to the crude particulate fraction has also been noted in other tissues [24,16]. It has been observed that addition of various agents, including several types of lipids, to avian or mammalian cells can stimulate cell fusion. Lucy [3] and Ahkong et al. [25] have used lysolecithin, unsaturated fatty acids and monoglycerides to induce fusion of avian eythrocytes. The agents that stimulated fusion, however, also led to considerable cell damage or lysis. More recently, Papahadjopoulos et al. [26] found that certain negatively charged, relatively fluid phospholipid vesicles were able to induce fusion of mouse and hamster cell lines without cytotoxicity. Lucy, Ahkong et al. and Papahadjopou10s et al. proposed that membrane fusion is highly dependent on membrane “fluidity” as well as on surface charge. The present studies indicated no significant change in the overall “fluidity” of the membrane as reflected by the cholesterol content and the total fatty acid composition. Neither the degree of unsaturation nor the chain lengths of the plasma membrane fatty acids changed during fusion. The contribution of the phospholipids to the net surface charge also did not vary during fusion, although other components of the membrane, e.g. glycolipids and proteins, especially glycoproteins, can also contribute to the surface charge. We are. therefore, presently examining total protein, glycoprotein and glycolipid compositions of the plasma membranes. The lack of significant changes in the gross lipid compositions of the plasma membranes was somewhat surprising in view of the dramatic morphological changes that take place during fusion. On the other hand, it is possible that highly localized changes in lipid composition are sufficient for fusion to begin and that these changes are too small to be detected in the gross lipid compositions reported here. In order to be able to detect such subtle changes, the fatty acid composition of each phospholipid class and turnover of the individual phospholipids are being investigated. In addition we are looking for a fusion-related phospholipase activity in the plasma membranes. The detergent action of the lysophospholipid product of such a phospholipase could cause drastic localized perturbations in membrane structure which could facilitate membrane fusion. Acknowledgments The authors thank Dr Richard Bischoff for helpful discussion and advice and Dr Jack Ladenson for performing the calcium determinations. The authors gratefully acknowledge the excellent technical assistance of MS Nancy Raglan. This work was supported in part by NIH Grant HL10406-08 and NSF Grant GB-38676X. C.K. and S.D.S. are the recipients of American Cancer Society Postdoctoral Fellowships PF-787 and PF-689, respectively.

321

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