Alterations in iodinated cell surface proteins during myogenesis

Preliminary

in lymphocytes. There have been previous reports of changes in the phosphorylation of nuclear [IO-121 and extranuclear [ 131proteins after mitogenic stimulation of lymphocytes. In most of these studies the changes occurred within the first few minutes after application of the mitogenic stimulus and then disappeared. The actions of lymphocyte mitogens can be effectively reversed, however, several hours after the initial application of the mitogen [14]. Biochemical reactions which occur several hours after the addition of mitogen but prior to the onset of DNA synthesis may therefore have regulatory significance. In only one study [ 121,was increased chromatin protein phosphorylation noted at the time of DNA synthesis. The time at which mitogenic stimulation becomes irreversible (10-12 h) is that time when increased PRP phosphorylation in extracts from ConA-treated cells was first detectable. Enhanced phosphorylation of PRP may be associated with an irreversible commitment to proliferation. It is clear that PRP is unique among soluble proteins in that its phosphorylatability in extracts from tumors [2], fetal and adult tissues [2], cultured cells [9], and lymphocytes is correlated with cell proliferation. We thank Carolyn E. McSwigan and Cathy Marquardt for their excellent technical assistance. This work was supported by a National Foundation-Basil O’Connor Grant, USPHS grants GM 22492 and CA 16228, and a National Leukemia Association grant.

notes

445

Casnellie, J E, Haley, B E & Greengard, P, Metabolism 24 (1975) 331. 7. Ashby, C D & Walsh, D A, Methods in enzymology (ed J G Hardman & B W O’Malley) vol. 38, p. 350. Academic Press, New York (1974). 8. Cross, M W & Ord, M G, Biochem j 124 (1971) 241. 9. Wehner, J M, Sheppard, J R & Malkinson, A M, Nature 266 (1977) 842. 10. Kleinsmith, L J, Allfrey, V G & Mirsky, A E, Proc natl acad sci US 55 (1%6) 1182. 118(1970) 191. Il. Cross,ME&Ord,MG,Biochemj 12. Johnson, E M, Kam, G & Allfrey, V G, J biol them 249 (1974) 4990. 13. Wedner, H J & Parker, C W, Biochem biophys res commun 62 (1975) 808. 14. Powell, A E & Leon, M A, Exp cell res 62 (1970) 315. Received August 16, 1977 Revised version received December 13, 1977 Accepted December 15, 1977

Alterations in iodinated cell surface proteins during myogenesis MARTIN MOSS, JAMES S. NORRIS, ERNEST J. PECK, JR and ROBERT J. SCHWARTZ, Department of Cell Biology, Baylor Houston, TX 77030, USA

College

of Medicine,

Lactoperoxidase catalysed iodination was used to label surface proteins of chick embryo muscle cells during myogenesis. Both quantitative and qualitative changes were observed between 1*51-labelledsurface proteins of pre-fusion, mid-fusion, and post-fusion cells. Significantly, two bands at 245000 molecular weight were present at pre-fusion but were observed as a single band at mid- and post-fusion. Radioactivity in this band increased selectively at post-fusion with a concomitant increase in lower molecular weight labelled proteins.

Summary.

Multinucleated muscle fiber formation occurs through the fusion of mononucleated myoblasts. Myoblast fusion has been shown References to be specific for skeletal muscle cells. 1. McCarty, K S & McCarty, K S Jr, J natl cancer Other cell types such as heart and kidney inst 53 (1974) 1509. 2. Malkinson, A M & McSwigan, C E, Biochem j. In [l] or fibroblast and liver [2] do not enter press. myotubes when mixed in fusing muscle cul3. Malkinson. A M. McSwigan. C E. Wehner. J M, Wang, T, Foker, J & Ha&w&ger, B W, Fed proc tures. Recently attention has been focused 36 (1977) 929. on the composition of the myoblast mem4. Wane. T. Marauardt. C & Foker. J. Nature 261 brane during fusion and myogenesis [3-S]. (i976)702. ' 5. Lowry, 0 H, Rosebrough, N J, Farr, A C & Since muscle cells and fibroblasts are found Randall, R J, J biol them 193(1951) 265. 6. Malkinson, A M, Krueger, B K, Rudolph, S A, together in primary cultures of embryonic Exp Cd Res I13 (1978)

446

Preliminary

notes

cible changes occurring in the sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoretic profiles of 1251-labelledcell surface proteins during myogenesis. Materials

woo-

b

,a

c

Fig. 1. ‘251-labelledmuscle cell surface proteins. Autoradiograph of SDS-polyacrylamide gel of (a) prefusion; (b) midfusion; (c) postfusion ‘2SI-labelled membrane

components. Muscle cell proteins were prepared and electrophoresed as described in Methods.

muscle, the examination of myoblast membranes necessitates preparation of relatively pure myoblast cultures. We have used a modification of the preplating technique of Yaffe [9] which is reported to selectively remove fibroblasts based upon their ability to attach to a plastic culture dish more rapidly than myoblasts. By performing three serial preplatings, we have obtained primary cultures of embryonic chick leg muscle which are highly enriched for myoblasts (greater than 90 %). Lactoperoxidase catalysed iodination was used to label muscle cell surface proteins. We report our observation of specific, reproduExp

Cell

Res

I13 (1978)

and Methods

Cell culture. Primary cultures of trypsinized 12-day chick embryo thigh scle were prepared by a modification of the met t?od of Konigsberg [lo]. Initial plating density was 10’ cells/l00 mm culture dish and after 3 serial preplatings was reduced to approx. 3 x lo6 cells/l00 mm dish. Culture medium consisted of 10% horse serum (Gibco), 1% penicillin-streptomycin, 0.25% Fungizone and 2.5% chick embryo extract in Dulbecco’s modified Eagle medium. Cultures were fed on the second day after plating and on every other day thereafter. Preplating was carried out on uncoated 100 mm culture dishes and cells were tinally plated onto gelatin coated dishes prepared as described by Hauschka [ 111. At 50 h postplating, 10-s M cytosine arabinoside (Sigma) was added to the cultures in order to selectively kill remaining Iibroblasts [ 121.Thfs was removed 48 h later by a medium change. We found that myoblasts underwent cell division for the first 38-40 h of culture and began fusion around 40 h. By 60 h, the majority of cells were fused myotubes. This observation is in agreement with other reports [13]. Because of this timetable, we designated prefusion cells 20 h, midfusion 45 h, and postfusion 6 days postplating, respectively. Label@ of surface proteins. Culture medium was removed by aspiration and dishes were washed 3 times with phosphate-buffered saline (0.9% NaCl, 0.01 M NaPO, plus 1% phenylmethyl sulfonylfluoride). Two ml of this solution was then added to each 100mm dish followed by 200 ~1 of a solution of lactoperoxidase bound to Sepharose beads (Worthington). Enzyme activity was 300 U/g of dry weight. Four hundred &i/ml of lZ51(New England Nuclear) was added followed by 20 PI/ml of a 1 : 1000 dilution of HzOz. The reaction was carried out at room temperature and stopped after 20 min by removal of the reaction mixture. Each culture dish was then washed 3 times with phosphatebuffered saline plus 0.01 M KI. Labelled cells were lysed with extraction buffer (5 % SDS, 8 M urea, 1% B mercaptoethanol, 0.05 M Tris-HCI, pH 7.0, and 1% phenylmethyl sulfonylfluoride), scraped with a rubber policeman, and passed through a 22 gauge syringe needle to shear DNA. The lysates were dialysed overnight against extraction buffer which was 1% in SDS. Dishes of cells containing all labelling reagents except lactoperoxidase were incubated for the normal reaction time and were not found to incorporate *251-label. Polyacrylamide gel electrophoresis. Aliquots of dialysed ‘2JI-labelled lysate were trichloracetic acid (TCA) precipitated onto glass fiber filters (Whatman) and counted for radioactivity. Approximately equal amounts of labelled protein (200000 cpm) were applied to separate slots of a 25 cm x 16 cm SDS polyacrylamide slab gel prepared according to Ames [14]. The body of this gel was 10% in acrylamide with a bisacrylamide to acrylamide ratio of 1 : 300, while the buffer system was identical to Laemmli [IS]. Molecular

Preliminary

notes

447

Fig.’ 2. Abscissa:

distance migrated (cm); ordinate: rel. absorbance. ---, Prefusion; -, midfusion (top); postfusion (botrom). 1251-labelledsurface protein profiles. Densitometric

scans of SDS polyacrylamide gel autoradiographs of prefusion vs midfusion (fop) and prefusion vs postfusion (bottom) patterns such as those shown in fig. I.

weight standards (fatty acid synthetase, 220000; myosin, 200000; bovine serum albumin, 68000; chymotrypsin, 22 500; myoglobin, 17500) and muscle culture samples were overlayed in 0.02% bromphenol blue and 10% sucrose and electrophoresed for 6 h at 35 mA. Gels were stained in 0.1% Coomassie brilliant blue, 50% TCA for 4 h. Destaining was carried out in a solution which was 30% methanol and 7 % acetic acid. Destained gels were dried down for autoradiography on Kodak XR-I Royal X-O Mat film at -70°C. Photographs of the exposed X-ray film were scanned in a Quick Scan densitometer. After autoradiography, each track of the gel was cut into 5 mm strips and counted in a gamma counter.

change is even more distinct at postfusion (fig. 2, bottom). Thus, it appears that M2 (230000 M,.), M, (210000 M,), M, (190000 M,.), M5 (180000 M,), and M, (140000 M,) are reduced while MI0 (30000 M,.), M,, (24000 M,), and M,, (21000 M,) are enhanced during this time. The most striking change was demonstrated by the presence of a 245000 M, double band at prefusion (MIA-MIB) which appeared to be replaced

Results

Autoradiographs of 1251-labelledchick muscle cell surface proteins are shown in fig. 1 to contain a number of proteins with a broad molecular weight distribution of 24500010500. The densitometric scans show more clearly the labelled protein profiles (fig. 2). Iodinated protein bands at 72000, 58000, 43 000, 30000 and 10500 M, were predominant at all stages of muscle cell development. A shift in labelled protein species was observed as the cells passed from prefusion to midfusion (fig. 2, top). This ?9t-781812

MIB ‘MIA

0

b

C

Fig. 3. Autoradiograph of 1251-labelled245000 M, pro-

tein change during myogenesis. Autoradiograph of SDS-polyacrylamide gel of (a) prefusion; (b) midfusion; (c) postfusion ‘*SI-labelled membrane components. Muscle cell proteins were prepared and electrophoresed as described in Methods. Equal counts were not applied to each slot of this gel. Exp Cell Rrs 113 (1978)

448

Preliminary

Table 1. Normalized

notes changes in cell surface proteins as measured by iodination % Differenceb Midfusion vs

Postfusion vs

rfusion

bTfusion

3.1 1.0 1.0 I.1

+41 +6 -7 i-7

+82 -33 -29 -15

1.7 1.8 3.1 4.5 2.6 1.9

1.2 2.4 2.9 1.9 1.9

-6 -5 -22 -10 -13 +0

-33 -37 -40 -42 -36 +0

1.2

1.2 1.5

1.8 1.5

+0 +25

+50 +25

5.6

8.4

+50

+96

% Total radioactivity’ Band

Apparent M,

Prefusion

Midfusion

M, M* M3 M*

245 000 230 000 210000 190000

1.7’ 1.5 1.4 1.3

2.4 1.6 1.3 1.4

2 4 Ml3 MS Ml0

180 000 140 72 000 58 000 43 ooo 30000

1.8 1.9 4.0 5.0 3.0 1.9

21 24 000 10 500

;I, lO”SO0 bandd

Postfusion

11

u Percent of total radioactivity was determined by assaying gel slices in distilled water in a -y counter. b Percent difference calculated as follows: Midfusion or postfusion % total radioactivity-prefusion prefusion % total radioactivity

% total radioactivity x loo

’ This number represents the total of MIA and M,B. d This band was shown to contain both TCA-precipitable and proteinase K-sensitive material. Gel slices at or ahead of the dye front did not contain either TCA precipitable or proteinase K-sensitive material and therefore were not included in total protein radioactivity.

by a single band (M,) at mid- and postfusion (figs 1 and 3). The two bands were also seen in the gel scans (fig. 2). However, these results must be viewed only as qualitative changes since the densitometric scans did not take into account the radioactivity in the 10500 M, band which was off scale. In order to present a quantitative assessment of iodinated protein changes during myogenesis the radioactivity of each gel slice was normalized as a percent of total iodinated material in the gel track (table 1). Incorporation in the M, band was observed to increase by 41% at midfusion and further increased at postfusion to a level about 82 % higher than that found at prefusion. The qualitative decreases observed in the gel scans (fig. 2) were also substantiated by the data in table 1. Bands Mz, M,, M, and M, were found to decrease by about 30% of E.rp Cc4 Res II3 (1978)

total radioactivity at postfusion. Individual decrements of at least 36 % of total radioactivity were observed in M, (72000 Mr), M, (58000 M,), and Mg (43 000 M,) although they were still among the most abundant proteins. The only band which was found to remain unchanged at all developmental stages was M,,. Postfusion incorporation into Ml1 and M,, was found to increase by about 25 and 50 %, respectively. The largest increase in incorporation was seen in the 10500 M,. band which increased by 50 % at midfusion and 96% at postfusion as compared to prefusion values. Midfusion levels of M1, M,, MS, Mg, M,, and the 10500 M, band were observed to approach final postfusion incorporation which suggests that these may be the earliest changes to occur. Midfusion radioactivity in Mz, MI, MS, Me, and M,, appeared close to prefusion values,

Preliminary

notes

449

tion of rat myoblasts [7] and fibroblasts [ 17, 181 as well as surface proteins of mutant cells deficient in substrate adhesiveness [ 191and protease sensitive surface proteins [20]. While none of these studies dealt directly with chick muscle, it is interesting to note that Hynes et al. [7] reported the only detectable difference in 1251-labelledcloned 68K Mr-, rat muscle cells as an enhancement in the large external transformation sensitive protein (LETS) after fusion. This protein is one of considerable interest and study in myoblasts and tibroblasts as it is seen to be very sensitive to proteolysis and is not present in the iodination profile of transformed cells. Our observation of an increase in lz51labelled LETS (corresponding to M,) is in agreement with Hynes; in addition, we were able to detect other changes upon Fig. 4. 1251-labelledproteins in culture medium. Proteins were labelled and electrophoresed as described muscle fusion. in Methods. Arrow corresponds to 68 000 M,. Because of the problem of fibroblast contamination, detailed studies of chick myoblast membranes have not been as numerwhile postfusion incorporation in these ous as those with cloned muscle cells [9, bands demonstrated major quantitative 211. With the selective removal of tibrochanges. blasts via preplating and subsequent treatIn order to demonstrate that the observed ment of cultures with cytosine arabinoside, pattern in iodination was not due to serum we have obviated the problem of major protein contaminants, tissue culture mefibroblast contamination. Furthermore, we dium was iodinated and fractionated by have succeeded in enhancing the resolution electrophoresis on a SDS polyacrylamide of the analytic system manyfold. This has gel (fig. 4). Serum proteins were intenbeen accomplished in part by the use of low tionally overloaded in order to detect any bis gels. These gels allow resolution of a minor bands that might be present. The broader range of molecular weights while major iodinated protein was found to run at greatly enhancing high molecular weight about 68000 M,. which corresponds to reband resolution [14]. In addition, the use of ported values for horse serum albumin [ 161. lactoperoxidase immobilized on Sepharose Thus, the iodinated bands observed in figs 1 beads insures that only exposed surface and 3 did not represent any serum contaproteins are labelled. mination. Our data suggest that a major structural Discussion reorganization of surface membrane protein Iodination of surface proteins catalysed by occurs in association with fusion (table 1). lactoperoxidase has been used to study sur- One possible cause of a redistribution of face changes associated with transforma- membrane proteins may be the calciumExp Cell Res 113 (1978)

450

Preliminary

notes

induced phase separation of phospholipids which is speculated to occur during fusion [22]. Recently, Chen was able to demonstrate a major redistribution of the LETS protein during and after fusion by immunofluorescence. He reported that when fusion was blocked, the redistribution of LETS did not occur. Furthermore, recognition of LETS was significantly reduced after fusion as determined by radioimmunoassay. Our observation of the 245000 iodinated material which appeared as a double band (M,A-M,B) at prefusion and a single band (M,) at mid- and postfusion might reflect specific modification or replacement of these surface proteins which may be necessary in order for fusion to occur. Modification of cell surface proteins appears to be important for cell-cell interaction in other systems. Hynes [lS] reported that proteolysis may reveal cryptic sites in the membranes of normal cells much like those seen in transformed cells. Pouyssegur & Pastan [ 191 observed that mutant fibroblasts with substrate adhesion deficiencies lacked many of the iodinated surface proteins found on nonmutant fibroblasts. Rutishauser et al. [23] proposed a model in which specific proteolytic cleavage of surface proteins may be involved in recognition and adhesion of embryonic chick neural retinal cells. Therefore, the observed increase in iodinated material in the 10500 M, band (table 1) may be suggestive of an increase in proteolytic activity associated with fusing muscle cells. It is tempting to speculate that specific proteolytic cleavage sites might exist on prefusion myoblasts which are modified in order to bring about fusion. Substantiation of increased protease activity associated with fusing muscle cells remains to be conclusively demonstrated. This possibility is currently under investigation in our laboratory. E-v Cell Res I I3 (1978)

This research was supported by the Muscular Dystrophy Association of America.

References 1. Yaffe, D & Feldman, M, Dev biol 11 (1965) 300. 2. Holtzer, H, Bischoff, R & Chacko, S, Cellular recognition, pp. 19-25. Appleton, New York (1969). 3. Reporter, M & Norris, G, Differentiation 1 (1973) 83. 4. Reporter, M & Raveed, D, Science lSl(1973) 863. 5. Kent, C, Schimmel, S & Vagelos, P, Biochim biophys acta 360 (1974) 312. 6. Winand, R & Luzzati, D, Biochimie 57 (1975) 764. 7. Hynes, R 0, Martin, G S, Shearer, M, Critchley, D R & Epstein, C J, Dev biol48 (1976) 35. 8. Chen, LB, Cell 10 (1977) 393. 9. Yaffe, D, Proc natl acad sci US 61 (1968) 477. IO. Konigsberg, I R, Methods in developmental biology, pp. 520-521. T. Y. Crowell Co., New York (1967). Il. Hauschka, S D, Growth, nutrition and metabolism of cells in culture, pp. 84-85. Academic Press, New York (1972). 12. Fishbach. G D. Dev biol28 (1972) 407. 13. Moss, P & Strdhman, R, Ceil 10 (1977) 265. 14. Ames Co. Catalog on disc electrophoresis, p. 14. Elkhart, Indiana (1976). 15. Laemmli, U D, Nature 227 (1970) 680. 16. Eisen, H N, Immunology, p. 375. Harper & Row, New York (1974). 17. Hogg, N M, Proc natl acad sci US 71 (1974) 489. 18. Hynes, R 0, Proc natl acad sci US 70 (1973) 3170. 19. Pouyssegur, J M & Pastan, I, Proc natl acad sci US 73 (1976) 544. 20. Teng, N & Chen, LB, Nature 259 (1976) 578. 21. Richler, C & Yaffe, D, Dev bio123 (1970) 1. 22. Weidekamm, E, Schudt, C & Brdiczka, D, Biochim biophys acta 433 (1976) 169. 23. Rutishauser, U, Thiery, J P, Brackenbury, R, Amisela, B & Edelman, G, Proc natl acad sci US 73 (1976) 577. Received August 18, 1977 Revised version received January 4, 1978 Accepted January 20, 1978

Sister chromatid viral disease

exchange in patients with

KAREN KURVINK,’ CLARA D. BLOOMFIELD* and JAROSLAV CERVENKA,’ ‘Divisions of Oral Patholoav and of Human and Oral Genetics. and zDepartment of kedicine, University of Minnesota Health Sciences Center. Minneauolis. MN 55455. USA

Sister chromatid exchange (SCE) frequencies were studied in differentially stained chromosomes from lymphocytes of 17 patients with viral disease. The mean SCE score for the patients was 8.7+

Summary.