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Differentiation of Cell Membranes in Cultures of Embryonic Chick Breast Muscle (Acetylcholine Receptor/Acetylcholinesterase/Adenylate Cyclase/Cell Fusion) JOAV M

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Differentiation of Cell Membranes in Cultures of Embryonic Chick Breast Muscle (Acetylcholine Receptor/Acetylcholinesterase/Adenylate Cyclase/Cell Fusion) JOAV M Proc. Nat. Acad. Sci. USA Vol. 71, No. 8, pp. 3208-3211, August 1974 Differentiation of Cell Membranes in Cultures of Embryonic Chick Breast Muscle (acetylcholine receptor/acetylcholinesterase/adenylate cyclase/cell fusion) JOAV M. PRIVES* AND BRUCE M. PATERSONt Departments of *Biophysics and tCell Biology, Weizmann Institute of Science, Rehovot, Israel Communicated by David Nachmansohn, June 6, 1974 ABSTRACT Three components of differentiated mus- Assays. The acetylcholine receptor was measured by the cle membrane, the acetylcholine receptor, acetylcholin- esterase (EC 3.1.1.7; acetylcholine hydrolase), and adenyl- binding of lnI-labeled a-bungarotoxin and is expressed as ate cyclase [EC 4.6.1.1; ATP pyrophosphate-lyase (cy- specific labeling (6). clizing)], appear simultaneously during myogenesis in Acetylcholinesterase was assayed by monitoring the hydrol- cultures of embryonic chick muscle, after the main period ysis of [1-14C]acetylcholine (Amersham) as described by of rapid cell fusion. However, unlike the cytoplasmic pro- Nirenberg and coworkers (11, 12). For each determination, teins of differentiated musele, the elaboration of these membrane components is unaltered when fusion is blocked tissue was removed by scraping from two 60-mm diameter by lowering the calcium concentration in the medium. culture dishes after three washes with Dulbecco's phosphate- These results suggest that membrane differentiation and buffered saline (13) and was stored as a pellet in 0.5 ml of cytoplasmic differentiation are regulated independently phosphate-buffered saline at -80°. Thawed samples were during muscle development. dispersed by sonication before assay. The final reaction mixture The differentiation of muscle in vitro involves the elaboration (11) contained 0-200,g of protein in a volume of 0.05 ml. of several specialized membrane components, including the Adenylate cyclase was assayed (14) with a reaction mixture acetylcholine receptor (1-3) and acetylcholinesterase (EC containing 40 mM Trise HCl (pH 7.4), 4 mM MgCl2, 2.2 mM 3.1.1.7; acetylcholine hydrolase) (4, 5), in the absence of direct theophylline, 2.7 mM [a-32P]ATP (1 mCi/mmol, Amersham), neuronal influence. We previously reported that the appear- 2.2 mM creatine phosphate, 30 /g of creatine kinase, and 10 ance of the acetylcholine receptor occurs subsequent to cell mM NaF. For each determination, four culture dishes 100- fusion in differentiating cultures of chick skeletal muscle; mm in diameter were rinsed with saline and scraped; the tissue however, if fusion is prevented by lowering the calcium con- was stored at -80° in 1 ml of 50 mM Tris* HCl (pH 7.6) until centration in the medium, the rate and extent of receptor use. Aliquots (50,Ml each; 500-800,gg of protein) of tissue were appearance are unaffected (6). In contrast, the cytoplasmic added per tube to initiate the reaction, which was performed proteins characteristic of differentiated muscle, such as in a final volume of 0.15 ml. Samples were incubated at 370 for myosin (7) and creatine phosphokinase (8, 9), are not syn- 20 min, and the reaction was terminated by boiling for 3 min. thesized in fusion-arrested cells. To each tube were added 101 cpm of [3H]cyclic AMP (New We now present evidence that two other components of England Nuclear Corp.) in 0.1 ml of 50 mM Tris- HCl (pH differentiated muscle cell membranes, acetylcholinesterase and 7.6) to allow correction for loss of cyclic AMP in the subse- adenylate cyclase [EC 4.6.1.1; ATP pyrophosphate-lyase quent procedure. Samples were diluted with 50 mM Tris HCl (cyclizing)], appear concurrently with the acetylcholine re- (pH 7.6) to a final volume of 1 ml and passed through columns ceptor in both control and fusion-arrested cultures. The of alumina (15) (0.5 X 2 cm) that had been equilibrated with simultaneous appearance of these constituents suggests that the same buffer. The columns were washed with 2.5 ml of the the differentiation of skeletal muscle cell membranes involves Tris buffer and the combined eluate (3.5 ml) was collected the coordinated synthesis of discrete membrane components directly into vials containing 15 ml of Triton-toluene scin- and is regulated independently of cytoplasmic differentiation. tillant [5.5 g of diphenyloxazole (PPO), 0.1 g of 1,4-bis[2-(4- methyl-5-phenyloxazolyl) ]-benzene (POPOP), 667 ml of METHODS toluene, and 333 ml of Triton X-100]. Cultures. Primary cultures of embryonic chick breast RESULTS muscle were prepared as described (6) and plated at initial densities of 2 X 106 cells per 60-mm dish and 6 X 106 cells per Appearance ofAcetylcholine Receptor and Acetylcholinesterase 100-mm dish. The cultures were grown in 88% (v/v) Dul- in Fusing and Fusion-Arrested Cells. The acetylcholine re- becco's'modified Eagle's medium (10) with 10% (v/v) horse ceptor appears 5-10 hr after the main period of fusion in con- serum (Gibco) and 2% (v/v) chick embryo extract. trol cultures, and the time and extent of receptor elaboration Where specified, growth medium was replaced by low cal- are unaffected when fusion is blocked by lowering the Ca++ cium medium (25juM calcium) prepared with Chelex 100 (6). concentration in the medium (Fig. 1). Cell fusion was scored as described (6). Cholinesterase activity was demonstrated in avian muscle by Nachmansohn (16), and this activity has been recently re- Abbreviations: BW284C51, 1,5-bis-(4-allyldimethylammonium- ported to increase in parallel with receptor in embryonic chick phenyl)pentane-1,3-dibromide; iso-OMPA, tetraisopropylpyro- muscle during development in ovo (17). The rapid kinetics phosphoramide. shown by the chick muscle cultures with respect to cell fusion 3208 Downloaded by guest on September 25, 2021 Proc. Nat. Acad. Sci. USA 71 (1974) Membrane Differentiation in Muscle Cultures 3209 70 I I I I I 1 I A 60F 12155 - 50- E 0 0 100 E 40O c 0 c 0 La) 2 75 -R 30- 0 ID 0 . 50 201 0 0 10 20 40 b60 80 1i00 Hours in vitro I r 20 40 60 80 100 HOURS IN VITRO r FIG. 2. Cholinesterase activity in control and fusion-arrested cultures. Results are expressed as the rates of hydrolysis of [14C]- BIB I I I I 55 acetyicholinesterase by control (0) and fusion-arrested (0) cultures. c D and receptor elaboration allow a precise comparison of changes 0 4z in activity patterns of acetylcholine receptor and acetyl- 0 cholinesterase with differentiation. In both control cultures and those in which fusion has been blocked, levels of cholin- E esterase activity increase rapidly and with similar kinetics be- x tween 40 and 70hr after plating (Fig. 2). a To determine the proportion of acetylcholine hydrolysis due Ct 3 .s to acetylcholinesterase, we assayed 50-, 60-, and 70-hr cultures 0 in the presence of one of two inhibitors, 10 1M 1,5-bis-(4- L- allyldimethylammoniumphenyl)pentane-1,3-dibromide (BW- 284C51, Burroughs, Wellcome and Co.), which specifically 0 blocks acetylcholinesterase (18), and 10 uM tetraisopropyl- . 0 pyrophosphoramide (iso-OMPA, Koch-Light Labs.), a specific inhibitor of cholinesterase (EC 3.1.1.8) (18). As shown in Fig. 3, BW284C51 inhibited acetylcholine hydrolysis by more than 75% in all cases, while iso-OMPA inhibited hydrolysis by less L than 20%. These data indicate that the major proportion of 20 40 60 80 100 acetylcholine hydrolysis, about 80%, is specifically due to Hours in vitro acetylcholinesterase, in agreement with earlier work (5), and FIG. 1. Kinetics of cell fusion and receptor appearance in that this proportion does not vary during the period of rapid control and fusion-arrested cultures. Plating density, 2 X 106 elaboration of acetylcholinesterase. cells per dish. (A) Fusion in control cultures (@) and in cultures changed to low calcium medium (25 MM Ca++) 24 hr after plating Acetylcholinesterase appears in parallel with the acetyl- (0). Fusion is expressed as the percentage of nuclei found in choline receptor in both control and fusion-arrested cultures. multinucleated cells. (B) Appearance of acetylcholine receptor This result is emphasized when the relative rates of appearance measured by specific binding of 125I-labeled a-bungarotoxin to of these two components are measured in the same cultures control (-) and fusion-arrested (0) cultures. Cells were incubated (Fig. 4), and are seen to be almost identical, suggesting that with 12 nM 1251-labeled a-bungarotoxin as described (6). The these functionally related proteins are synthesized in a co- specific activity of the toxin used in these determinations was ordinated manner. about 1010 cpm/lumole. Toxin bound at 85 hr corresponded to about 5 X 10-13 moles per culture or 4.5 X 10-13 moles/mg of Appearance of Adenylate Cyclase. It was of interest to con- protein. trast the kinetics of appearance of acetylcholine receptor and Downloaded by guest on September 25, 2021 3210 Biochemistry: Prives and Paterson Proc. Nat. Acad. Sci. USA 71 (1974) c 120 *110 E C E 7.5 E 90- 0 E E0- 0 E 5.0 l0- B 0 A 60 a. 50 25 =C 0 40 .430- < 20- I I I I 20 40 60 80 100 Hours in vitro FIG. 5. Appearance of adenylate cyclase activity in control °' (1) (2)(3 (1) (2) (3 (1) (2)(3) (0) and fusion-arrested cultures (0). Cells were plated at a FIG. 3. Effects of esterase inhibitors on rates of acetylcholin- density of 6 X 106 cells per 100-mm culture dish, and tissue from esterase hydrolysis by muscle cultures at (A) 50 hr, (B) 60 hr, four dishes was pooled for each determination. and (C) 70 hr after plating. Hydrolysis was measured in control cultures (1) and in the presence of 10 MM BW284C51 (2), and 10MM iso-OMPA (3).
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